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Trace elements present in airborne particulate matter—Stressors of plant metabolism
Ecotoxicology and Environmental Safety 79 (2012) 101–107
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Trace elements present in airborne particulate matter—Stressors of plant metabolism Milan Pavlı´k a, Daniela Pavlı´kova´ b,n, Veronika Zemanova´ b, Frantiˇsek Hnilicˇka c, Veronika Urbanova´ b, Jiˇrina Sza´kova´ b a
ˇ ´ 1083, 14220 Prague, Czech Republic Isotope Laboratory, Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Vı´denska ´ 129, ´cka Department of Agro-Environmental Chemistry and Plant Nutrition, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Kamy 16521 Prague, Czech Republic c ´ 129, 16521 Prague, Czech ´cka Department of Botany and Plant Physiology, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Kamy Republic b
a r t i c l e i n f o
abstract
Article history: Received 25 August 2011 Received in revised form 12 December 2011 Accepted 13 December 2011 Available online 31 December 2011
Changes of amino acid concentrations (glutamic acid, glutamine, asparagine, aspartate, proline, tryptophan, alanine, glycine, valine and serine), gas-exchange parameters (net photosynthetic rate, transpiration rate, stomatal conductance and intercellular CO2 concentration) and nitrate levels in Lactuca serriola L. under airborne particulate matter (PM) contamination reported here reveal their role in plant chronic stress adaptation. Results of the pot experiment confirmed the toxic effect of trace elements present in PM for lettuce. PM applied to soil or on the lettuce leaves were associated with the strong inhibition of above-ground biomass and with the enhancement of plant trace element contents. The significant changes of amino acid levels and leaf gas-exchange parameters of the plants showed strong linear dependences on PM contamination (R2 ¼ 0.60–0.99). PM application on leaves intensified toxic effect of trace elements (As, Pb, Cr and Cd) originating from PM by shading of the leaf surface. The plant accumulation of nitrate nitrogen after PM contamination confirmed to block nitrate assimilation. & 2011 Elsevier Inc. All rights reserved.
Keywords: Airborne particulate matter Amino acids Gas-exchange parameters Lactuca serriola L. Toxic elements
1. Introduction Air pollution is one of the most important environmental problems in densely populated and industrialized areas. Airborne particulate matter (PM) in urban air has been at the center of recent concerns, mainly due to its adverse health effects on the urban population. Among the characteristics of particulate matter that relate to its toxicity could be mentioned the presence of trace metals (Rodriguez et al., 2010). Motor vehicles have been demonstrated to be a major contributor of particle-bound trace metals (Valavanidis et al., 2006) in urban areas. As plants are immobile and more sensitive in terms of physiological reaction to the common air pollutants than humans and animals, they better reflect local conditions. High accumulation due to airborne particulate matter was found for Pb, Cr and Cd, especially in leafy vegetables (Voutsa et al., 1996). The major contribution of most trace elements to vegetable leaves was from atmosphere. According to De Temmerman and Hoenig (2004) the leaves accumulate the deposited airborne trace elements; however, they are also influenced by soilborne metals. Mechanisms of air pollution
n
Corresponding author. Fax: þ420 234381801. E-mail address: [email protected] (D. Pavlı´kova´).
0147-6513/$ - see front matter & 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2011.12.009
toxicity are very complex and depend on various physiological and biochemical properties of plants. The PM deposited on the leaf surface can affect the plant’s metabolism by blocking light, obstructing stomatal apertures, increasing leaf temperature and altering pigment and mineral contents of the leaf (Kuki et al., 2008). Surface dust deposits may alter the optical properties of leaves, particularly the surface reflectance in the visible and short wave infrared radiation range (Hope et al., 1991). In response to these adverse effects various biochemical changes also occur such as decreased chlorophyll content and increased ascorbic acid content of leaves. The significant negative correlation between dust load and leaf pigment content in vegetation near the highway was confirmed by Prusty et al. (2003). According to Poma et al. (2002) in plant tissues an enhancement of the specific activity of the stress-related enzyme peroxidase was monitored and were confirmed genotoxic activities associated with the coarse (PM 10 smaller than 10 mm) and the fine fraction (PM 2.5 smaller than 2.5 mm) of airborne particulates. Field transect studies have shown significant negative correlations between air pollutant concentrations and net photosynthesis, biomass accumulation and yield of crop plants (Agrawal, 2005). Plant stress metabolism has been usually studied in experiments focused on short-term effects of stress agens, i.e. in the form of an acute stress. This form of stress, however, does not
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reflect correctly the environmental conditions (di Toppi and Gabbrielli, 1999). Plants usually grow and develop in conditions of long-term chronic stresses—they take up small quantities of several toxicants after a long time period. For this reason, the PM study has been focused on complex changes of plant metabolism (plant growth, photosynthesis, N metabolism, etc.) under chronic stress. Wuytack et al. (2011) recommended the use of plant characteristics, such as growth, total biomass production and specific leaf area, as diagnostic monitoring tools. Amino acid metabolism may play an important role for plant growth and development as well as in plant stress resistance, by osmotic adjustment and the accumulation of compatible osmolytes, detoxification of active oxygen species and risk elements and intracellular pH regulation (Singh, 1999). We suppose that the impact of trace elements originated from airborne particulate matter on photosynthesis and nitrogen utilization by plants can result in changes of amino acids concentrations of leafy vegetable. The present study was conducted to examine the relationship between amino acids concentrations and leaf gas-exchange parameters as a result of lettuce contamination by trace elements contained in airborne particulate matter.
Table 2 Total concentrations of selected trace elements in tested soil and in airborne particulate matter determined at the beginning of experiment. The values represent the means of data obtained in the experiment (n ¼2, i.e. two experimental years). As mg kg Chernozem Airborne particulate matter
Cd
Cr
Pb
1
39.47 0.01 0.69 7 0.07 65.5 7 2.76 30.4 7 2.2 24.57 0.85 2.06 7 0.27 125.5 7 3.27 131.5 7 8.9
was dissolved in 20 mL of 1.5% HNO3 (v/v) (electronic grade purity, Analytika Ltd., Czech Republic) and kept in glass tubes until the analysis. Aliquots of the certified reference material RM NCS DC 73350 poplar leaves (purchased from Analytika, CZ) were mineralized under the same conditions for quality assurance. Total element concentrations in PM and in soil were determined in digests obtained by two-step decomposition as follows: 0.5 g of the sample was decomposed by dry ashing in a mixture of oxidizing gases (O2 þO3 þNOx) in an Apion Dry Mode Mineralizer (Tessek, CZ) at 400 1C for 10 h; the ash was then dissolved in a mixture of HNO3 þ HF (2:1 v/v), evaporated to dryness at 160 1C and dissolved in diluted Aqua Regia (Pavlı´kova´ et al., 2008). The concentrations of trace elements were determined by ICP-OES with axial plasma configuration (Varian VistaPro, Varian, Australia). The toxic effects of As, Cd, Cr and Pb for plant was evaluated for their highest concentration in PM (Table 2).
2. Materials and methods 2.1. Plant material and cultivation conditions Adaptation of lettuce (Lactuca serriola L. var. capitata cv. Detenicka atrakce) plants to trace elements present in air pollution was investigated in pot experiment repeated for two years. For this experiment, lettuce plants (seeds obtained from SEMO Ltd. Smrzˇice, Czech Republic) were planted into plastic pots containing the soil as specified below. The plants (one plant per pot) were cultivated from April to June under natural light and temperature conditions at the experimental hall of the Czech University of Life Sciences Prague, Czech Republic. The water regime was controlled and the soil moisture was kept at 60% maximum waterholding capacity. Airborne particulate matter using for experiment was sampled in air condition units in large buildings near highway (Prague, Czech Republic) with heavy traffic (87 thousand of motor vehicles per day). We got the dust mostly from vehicular traffic with a dominance of fine particles. The fraction o 0.065 mm was used for this experiment. For cultivation of lettuce plants, 5 kg of chernozem (pHKCl ¼7.2, Cox ¼ 1.83%, CEC¼ 258 mval kg 1) was thoroughly mixed with 0.5 g N, 0.16 g P and 0.4 g K applied in the form of ammonium nitrate and potassium hydrogen phosphate for all treatments. Lettuce was exposed to a PM applied to soil (the soil was spiked prior to the experiment with 30 g PM per pot) or to the application of suspended PM on the leaves during the vegetation (Table 1). Lettuce leaves were sprayed by suspended PM eight times (3.75 g PM 100 mL 1 per pot for every spraying) during 4 weeks. The soil contents of toxic elements were analyzed as total contents and the portions of available elements are very low in this soil therefore the effect of these contents on plants are not high. Each treatment was performed in five replications. Lettuce plants were planted up to the stage of full leaves development (75 days after planting). The lettuce leaves were washed up after harvest. The plants from each pot were analyzed individually. 2.2. Analyses 2.2.1. Analyses of trace elements Plant samples were decomposed using the dry ashing procedure as follows: an aliquot ( 1 g) of the dried and powdered biomass was weighed into a borosilicate glass test-tube and decomposed in a mixture of oxidizing gases (O2 þO3 þNOx) at 400 1C for 10 h in a Dry Mode Mineralizer Apion (Tessek, Czech Republic). The ash
2.2.2. Analysis of free amino acids The amino acids from methanol þH2O extracts were determined using EZfaast amino acid analysis procedure (Phenomenex, USA). Samples were analyzed for amino acid contents by GC–MS using the Hewlett Packard 6890N/5975 MSD (Agilent Technologies, USA). Samples were separated on a ZB-AAA 10 m 0.25 mm amino acid analysis GC column under these conditions: the carrier gas (He) flow was kept constant at 1.1 ml min 1. The oven temperature program was the following: initial temperature 110 1C, a 30 1C min 1 ramp to 320 1C. The temperature of the injection port was 280 1C. A 1.5–2 ml sample was injected in split mode (1:15, v/v). MS conditions were as follows: MS source 240 1C, MS quad 180 1C, auxiliary 310 1C, electron energy was 70 eV, scan m/z range 45–450 and sampling rate was 3.5 scan s 1 (Pavlı´k et al., 2010a). The complex of free amino acids was determined (alanine, glycine, valine, leucine, isoleucine, threonine, serine, tryptophan, proline, asparagine, aspartate, methionine, glutamic acid, glutamine and lysine). The concentrations of the free glutamine, lysine and methionine were below detection limit of GC. 2.2.3. Analysis of nitrate nitrogen in plant biomass Dried above ground biomass was extracted by hot distilled water (1:10, w/v). Contents of N–NO3 were determined using segmental flow-analysis using a colorimetric method on a SKALARplus SYSTEM (Skalar, Netherlands). 2.2.4. Determination of gas-exchange parameters The net photosynthetic rate (PN), transpiration rate (E), stomatal conductance (gs) and intercellular CO2 concentration (Ci) were measured in the leaves in situ using the portable gas-exchange system LCproþ (ADC BioScientific Ltd., Hoddesdon, Great Britain) from 10:00 to 11:30 of Central European summer time the both experimental years. The irradiance was 802–821 mmol m 2 s 1 photosynthetically active radiation (PAR), the temperature in the measurement chamber was 25–26.5 1C, the CO2 concentration was 550750 cm3 m 3, the air flow rate was 205730 mmol s 1 and the duration of the measurement of each sample was 15 min after the establishment of steady-state conditions inside the measurement chamber (Hola´ et al., 2010). From these data, the water use efficiency was estimated (WUE¼ PN/E). For calculation of linear correlation (R2) Statistica for Windows version 7.0 CZ was used (StatSoft, Inc., Tulsa, OK).
3. Results Table 1 Experimental design. Treatment
Fertilization
Particulate matter o 0.065 mm (30 g per pot)
1 2 3
NPK NPK NPK
0—control Soil Plant leaves
Results of the pot experiment revealed the toxic effect of PM for lettuce plants. Plant response to the PM contamination was assessed on the basis of a decreased lettuce leaves dry matter and increased concentrations of trace elements in the above-ground biomass (Table 3). The strong inhibition of above-ground biomass was observed on the treatment 3 (PM application on lettuce leaves). Compared to the untreated control, the biomass yield of
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Table 3 Dry matter yield and concentrations of selected trace elements in lettuce (Lactuca serriola L. var. capitata cv. Detenicka atrakce) above-ground biomass. The values represent the means of data obtained in the experiment (n ¼10, i.e. two experimental years and five replications per each year). Treatment
1—Control 2—PM soil 3—PM plant
Yield of dry matter
As
Cd
g Per pot
mg kg 1 Dry matter
17.8 7 3.6 14.9 7 4.8 8.1 7 1.6
0.637 0.26 1.007 0.07 1.247 0.78
0.877 0.20 1.447 0.43 0.817 0.39
Cr
Pb
N–NO3
0.247 0.01 0.337 0.13 1.147 0.48
1.657 0.49 1.877 0.82 2.457 1.70
116 78 222 720 345 72
Table 4 Effect of PM contamination on CO2 intracellular concentration (Ci), the net photosynthetic rate (PN), the stomatal conductance (gs) and transpiration rate (E) of lettuce (75th day from sowing). From these data, the water use efficiency was estimated (WUE¼ PN/E) (n ¼30, the values represent the means of data obtained in two experimental years). Treatment
Ci (vpm)
PN (mmol CO2 m 2 s 1)
gs (mol m 2 s 1)
E (mmol H2O m 2 s 1)
WUE
1—Control 2—PM soil 3—PM plant
336.827 9.56 346.227 12.43 387.467 12.71
11.31 7 1.25 7.46 7 1.05 2.75 7 1.44
0.927 0.26 0.617 0.08 0.267 0.14
3.04 70.64 3.78 70.32 5.73 70.89
3.72 0.16 0.05
treatments 3 was reduced to ca. 45% of control treatment. PM application to soil reduced the yield only by 16% in contrast to control treatment. The highest contents of trace elements (As, Cr and Pb) were determined on treatment after PM application on leaves. Compared to the untreated control, the As content in above-ground biomass was enhanced up to 2 fold, the Pb content 1.5 fold and the Cr content 4.8 fold. Cadmium content was not increased. The Cd content in plant biomass was determined in extent 0.8170.39 mg kg 1—the highest Cd content was 1.2 mg kg 1 (the control highest content 1.07) and difference between this treatment and control treatment is not significant. Our results showed the dominant effect of As, Pb and Cr on plant metabolism from PM applied on leaves compared to Cd. Its content in above-ground biomass was significantly affected by PM application to soil and it increases more than by 65% compared to control treatment. PM application to soil affected the As, Cr and Pb contents in above-ground biomass less than PM application on leaves. The As content in above-ground biomass was enhanced up by 58%, the Cr by 38% and Pb content by 13% compared to control treatment. The effect of soil conditions (soil organic matter, cation exchange capacity and clay content) to the trace elements adsorption was significant. The behaviors of trace elements from PM were studied in two different soil samples and results confirmed the effect of soil characteristics (the results have published not yet). According many results Cd was of a greater potential of entering the human food chain than Pb. The net photosynthetic rate (PN) has been examined together with the stomatal conductance (gs), transpiration rate (E) and intercellular CO2 concentration (Ci) as potential selection marker for the detection of plant stress. Under PM contamination, the changes in leaf gas-exchange parameters—PN and gs—were similarly directed: both decreased and were lower than in control treatment (Table 4). PM application on leaves resulted in strong decrease of of PN and gs by 72% and 76%, respectively, as compared to control treatment. Stomatal conductance typically declines in response to increasing intercellular CO2 concentration. This finding was confirmed by our results—Ci was increased after PM application on leaves by 15% compared to control treatment. Transpiration rate was increased with PM contamination (Table 4). PM application on lettuce leaves showed high rate of transpiration, higher by 88% than control treatment. The close relationship between leaf gas-exchange parameters and content of trace elements in plant biomass was confirmed using linear correlation (Table 5). The contents of As, Cr and Pb showed highly significant correlation (R2 ¼0.79–0.99), whereas the
Table 5 Linear correlation (R2) between leaf gas-exchange parameters and trace elements contents in above-ground biomass of lettuce. Leaf gas-exchange parameters
As
Cd
Cr
Pb
Ci (vpm) PN (mmol CO2 m 2 s 1) E (mmol H2O m 2 s 1) gs (mol m 2 s 1) N–NO3 content (mg N kg 1 dry matter)
0.79 0.97 0.87 0.97 0.97
0.18 0.02 0.11 0.02 0.02
0.99 0.87 0.97 0.85 0.86
0.99 0.96 1.00 0.96 0.96
correlation was not found between Cd and leaf gas-exchange parameters. Changes of gas-exchange parameters confirmed the decrease of C and N assimilation as result of PM contamination (Table 4). The nitrate levels of PM treatments were raised compared to the control (Table 3). The significant correlations between leaf N–NO3 and leaf gas-exchange parameters were confirmed (R2 ¼0.90–0.99). The contents of nitrate levels and As, Cr and Pb showed significant correlation (R2 ¼0.86–0.97). Nitrogen flow through the amino acids can change dramatically in response to stress. Glutamate (Glu), glutamine (Gln), aspartate (Asp) and asparigine (Asn) are used to transfer nitrogen from source organs to sink tissues and to build up reserves during periods of nitrogen availability for subsequent use in growth, defense and reproductive processes. The changes of free amino acid concentrations after PM application are demonstrated in Fig. 1. Glu and Asp are key amino acids for biosyntheses of nucleic acids, ATP, cytokinins and chlorophyll. Concentration of these amino acids showed a downward trend after PM application. PM application on leaves resulted in strong decrease of free Glu (by 75%) and Asp (by 78%) contents compared to control treatment (Table 6). Glu together with cysteine (Cys) are important for detoxification of trace elements via formation of phytochelatins. The level of free Gln was low to non detectable in the plant samples analyzed and did not change when the plants were treated with PM. Asn levels changed drastically under stress conditions. Its significant increase was observed after PM application on soil (by 98%) and mainly on leaves (298%). The accumulation of free Asn could be associated with the remobilization of assimilated nitrogen as proteins and other substance. The concentrations of these amino acids showed strong linear correlations with As, Cr and Pb contents in plant above-ground biomass (As R2 ¼0.90–0.99; Cr R2 ¼0.62–0.94; Pb R2 ¼0.79–99) (Table 7). The Cd effect is not significant (R2 ¼0.02–0.08).
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Anthocyans,
AMP, ADP, ATP
Ornithine
Arginine
Antranilate
Glutamate-5-semialdehyde
5-aminolevulinate
Lysine Aspartate-4-semialdehyde
Aspartate
Pyrimidines
Methionine
Ascorbate-glutathione cycle
Glycine
Alanine
ne
Homocysteine al o Ox
Valine
Chlorophyll sis t he syn ot o Ph
athio Glut
Homoserine
Isoleucine
Chorismate
Phytochelatine
Threonine
Nucleic acids
4-Hydroxyphenylpyruvate
Phenylpyruvate
Hydroxyproline
Asparagine
Aminobenzoate
Lignin Proline
Purinebases
Tyrosine
Phenylalanine
Ureacycle
Glutamine,Glycine, CO 2, N 10-formyltetrahydrofolate
Tocopherols Folate
Indol
Alcaloids,
Cytokinines
Tryptophan
Coumarins,
Furanocoumarins
Polyamines
Indole auxins
Flavonoids,
Tannins
Shikimate
Damage&protection mechanismofplant against reactiveoxygenspecies
Glucose
Serine
t eta ac
Pyruvate
AcetylCoA
e
Citratecycle
lu xog 2-O
tar
at e
Cysteine Glycerophospholipids Glutamate
Threonine
Glutamine
Leucine
Fig. 1. Metabolism of amino acids.
Table 6 Content of selected free amino acids in lettuce (Lactuca serriola L. var. capitata cv. Detenicka atrakce) above-ground biomass. The values represent the means of data obtained in the experiment (n¼ 10, i.e. two experimental years and five replications per each year). Amino acid (mmol kg 1)
Glu Asp Asn Pro Ala Phe Trp Gly Val Leu Ile Thr Se a
Treatments 1—Control
2—PM soil
3—PM plant
2430 7 42 2566 7 58 35.8 7 2.8 98.4 7 11.6 302 736 52.3 7 10.6 11.3 7 2.6 33.4 7 6.7
16087 28 1178 7 32 71.07 1.9 2237 48 2947 32 33.07 9.8 20.17 5.3 30.07 7.9 37.17 14.1 27.77 3.7 24.77 5.2 1637 17 3877 21
589 7 21 571 7 19 142 7 10.9 123 7 17 264 7 36 31.3 7 4.9 29.7 7 4.9 28.8 7 5.9 51.2 7 10.9 39.4 7 4.6 28.2 7 1.7 157 7 21 361 7 19
a
34.8 7 6.1 28.7 7 4.1 a a
Concentrations of free amino acids were below detection limit of GC.
The amino acids proline (Pro) and ß-alanine (Ala) accumulates markedly in response to stress in plants. The increase of free Pro content in aboveground biomass was detected in our experiment. The most significant increase of free Pro concentration was observed after PM application to soil (by 126%) in contrast to control treatment. The increase of Pro after PM application on lettuce leaves was only by 25% compared to control treatment. The linear correlation between Pro and Cd content in plant (R2 ¼0.93) and Pro increase after PM application to soil confirmed
Table 7 Linear correlation (R2) between the contents of free amino acids and trace elements contents or N–NO3 content in above-ground biomass of lettuce. Amino acid
As
Cd
Cr
Pb
N–NO3
Glu Asp Asn Pro Ala Phe Gly Val Leu Ile Thr Ser Trp
0.97 0.99 0.90 0.09 0.81 0.90 0.98 0.98 0.08 0.06 0.83 0.80 0.98
0.02 0.02 0.08 0.93 0.16 0.13 0.03 0.03 0.90 0.96 0.20 0.23 0.01
0.87 0.63 0.94 0.06 0.99 0.40 0.58 0.60 0.55 0.10 0.31 0.28 0.84
0.96 0.79 0.99 0.01 0.99 0.58 0.75 0.76 0.46 0.02 0.48 0.45 0.95
0.99 0.93 0.98 0.02 0.92 0.78 0.91 0.92 0.18 0.01 0.69 0.66 0.99
the Cd effect mainly after root uptake. An alanine decrease after PM application was not significant (only by 13%). Its content has strong linear correlations with As, Cr and Pb contents in contrast with Pro (Table 7). Fig. 1 shows that serine (Ser) is associated with formation of glycophospholipids and glutathione via Cys biosynthesis. Free Cys concentration was below detection limit of GC. Serine and glycine (Gly) are formed as intermediate products in the photorespiratory cycle. Phosphoglycolate formed by photorespiration is the precursor for their formation, and the latter cysteine is formed. The changes of Gly were not observed. Free serine concentration of plants growing on control treatment was below detection limit of GC. The both PM treatments showed increase of Ser concentration, but difference between them was not observed.
M. Pavlı´k et al. / Ecotoxicology and Environmental Safety 79 (2012) 101–107
The importance of the branched-chain amino acids – valine (Val), leucine (Leu) and isoleucine (Ile) – as building blocks of proteins in plants is obvious. The changes of free Leu and Ile concentrations were not significant. The content of Val increased dramatically, mainly after PM application on lettuce leaves. Tryptophan (Trp) plays a major role in the regulation of plant development and defense responses. Trp is the precursor for auxin producing plant. Our results showed the significant increase of this amino acid for both PM treatments, mainly after PM application on lettuce leaves (by 163%). The inhibition of plant growth was affected by changes of free Gln, Glu, Asp and Trp concentrations. These amino acids play an importance role in syntheses of active forms of auxin and cytokinines (Fig. 1). The significant correlations between leaf N– NO3 and these amino acids were confirmed—R2 ¼0.93–0.99 (Table 7). No significant differences in results of this experiment were observed between individual experimental years.
4. Discussion Plant response to the PM toxic effect for lettuce plants was assessed on the basis of a decreased lettuce leaves dry matter, increased concentrations of trace elements and nitrates in the above-ground biomass, the changes in leaf gas-exchange parameters and amino acid concentrations. Decreased yield of dry matter and increased concentrations of trace elements in the above-ground biomass were determined for both contaminated treatments. Our data correspond with those by Pavlı´kova´ et al. (2002, 2007, 2008) who reported that excessive amounts of toxic elements in contaminated soil inhibited plant growth and development due to their phytotoxicity. Reduced growth observed at contaminated treatments may be partly ascribed to lower net photosynthetic rate; but not exclusively, since it has been argued that the reduced growth might be also due to increased tissue permeability. It might also result from inhibition of cell division (Redondo-Go´mez et al., 2011). Thus, reduction in growth can be linked to the high trace elements accumulation, as in this case plants have to spend extra energy to cope with the high trace element concentrations in the tissues (Israr et al., 2006). The strong inhibition of above-ground biomass and the significant changes in leaf gas-exchange parameters and amino acid concentrations were determined on treatment after PM application on leaves. PM application on leaves affected plants: (1) by toxic effect of trace elements from PM and (2) by shading of the leaf surface. There were very clear effects of PM contamination on PN and gs in our experiment and the decrease of PN involved a parallel decline of water use efficiency at both PM treatments of the experiment. The changes determining after PM application on leaves were increased by the shading effect. According to Hirano et al. (1995) the dust decreased the photosynthetic rate by this effect and the dust of smaller particles had a greater shading effect. Hope et al. (1991) reported that surface dust deposits may alter the optical properties of leaves, particularly the surface reflectance in the visible and short wave infrared radiation range. The parallel change of PN and gs in lettuce leaves reinforce evidence that the changes in PN could be mainly attributed to the changes in gs (Chartzoulakis et al., 2002). The decrease of gs may be related to an alteration in the K:Ca ratio in the guard cells and/or alterations in the concentration of abscisic acid, which controls stomatal movement . According to Pandey and Sharma (2003) gs reduction in plants of Brassica oleracea treated with Cr3 þ
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was presented by decreases in water potential and transpiration rates, as well as increases in diffusive resistance. The positive correlations of PN, E and gs with the stomatal length in the upper epidermis and the lower epidermis thickness, as well as the negative correlation of PN with the lamina thickness and palisade tissue thickness were observed in Zn stressed plant by Shi and Cai (2009). Stomatal limitation is often considered as an early physiological response to stress, which results in decreased PN, through limited CO2 availability in the mesophyll (Cornic, 2000). However, there is strong evidence that photosynthetic processes in the mesophyll such as RuBisCo activity, ATP supply, electron transport rate and light capture efficiency in the photosystems become impaired as the stress increases (Vaillant et al., 2005). Therefore, if the limitation of PN in plant were due to gs, there would be a reduction in intercellular CO2 concentration. The increase of Ci may be explained by modifications of RuBisCO activities of plant (Redondo-Go´mez et al., 2011). The inhibition in enzyme activity in presence of trace elements could be due to substitution of Mg2 þ in the active site of RuBisCO or chlorophyll subunits by metal ions (Siedlecka and Krupa, 2004). This substitution leads to instability of chlorophyll molecule and to decrease of photosynthesis efficiency. Trace elements have been found to decrease the Chl content and the Chl a/b ratio in many terrestrial plants (Khudsar et al., 2004). These parameters were correlated with PN. The results of Shi and Cai (2009) suggest that PN inhibition could be caused by Chl content reduction. The significantly increased Ci was determined only after PM application on leaves. PM application to soil did not affect Ci. The significant shading effect on Ci was confirmed by this finding. According to Hermle et al. (2007) the CO2 concentration in the leaf intercellular air space was increased as responses of Populus tremula to trace element dust. Gas exchange responses of plant to trace elements were attributed to leaf structural and ultrastructural changes resulting from hypersensitive-response-like processes and accelerated mesophyll cell senescence and necroses in the lower epidermis, especially along the transport pathways of trace elements in the leaf lamina. Transpiration rate was increased with PM contamination in our experiment. PM application on lettuce leaves showed high rate of transpiration, higher by 88% than control treatment. Majority of authors published decrease of E as plant response to trace elements (Hermle et al., 2007; Shi and Cai, 2009 etc.). Moreover, Hirano et al. (1995) have found that the additional absorption of incident radiation by the dust increased the leaf temperature, and consequently changed the photosynthetic rate in accordance with its response curve to leaf temperature. The increase in leaf temperature also increased the transpiration rate. Under shaded conditions, carbon uptake per unit leaf biomass is lower than under full light conditions (Van Hess and Clerkx, 2003). According to Wuytack et al. (2011) the degree of shadow needs to be taken into account in biomonitoring studies. Nitrate is a major nitrogen source for plants. The most plants devote a significant portion of their carbon and energy reserves to its uptake and assimilation. The reduction in photosynthetic rate led to a limited supply of metabolic energy and therefore to N assimilation restriction. The accumulation of nitrate nitrogen after PM application on leaves has been shown to block nitrate assimilation. Robinson et al. (1998) have found that the nitrate level was raised while nitrate reductase activity declined heavily at the polluted site of leaf during different stages of plant growth, compared to the control. Nitrate reductase is a key enzyme of nitrogen metabolism. It catalyzes the first step in the assimilatory reduction of nitrate into ammonia, which reduces nitrate to nitrite. Inhibition of nitrate reductase activity by trace element
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stress has been reported in higher plants (Nagajyoti et al., 2010). The decrease in nitrate reductase activity in trace metal treated plants could also reflect a decrease in photosynthesis because sugars are essential for nitrate reductase expression (Solanki and Dhankhar, 2011). A linear dependence of photosynthetic capacity on leaf nitrogen content was assumed by Leuning et al. (1995). PM contamination caused depletion in the pools of free glutamate and asparagines—amino acids that are metabolized during photorespiration. Glutamate, glutamine, aspartate and asparagines are used to transfer nitrogen from source organs to sink tissues and to build up reserves during periods of nitrogen availability for subsequent use in growth, defense and reproductive processes. These compounds are the major amino acids translocated in the phloem of most species. Gln is not only the major amino acid used for nitrogen transport, but also a key metabolite that acts as an amino donor to other free amino acids, primarily catalyzed by glutamate synthase. This pathway interacts with carbohydrate metabolism or the energy status of the organ (Hodges et al., 2002). Asparagine accumulates under conditions of stress. There is evidence that asparagine can bind to cadmium, lead and zinc. Cadmium stress on Zea mays L. decreased the glutamine and asparagine content. The findings of Pavlı´k et al. (2010b) have confirmed these results and have showed significant decrease of asparagine concentration after arsenic stress on spinach. Lea et al. (2007) have published opposite results. Cadmium induced an almost 10-fold increase in the asparagine concentration of tomato roots but only fourfold in the leaves. Accumulation of free proline in plants has often been reported as a consequence of a wide range of environmental stress (Pavlı´kova´ et al., 2008). The increase of free proline content in aboveground biomass was detected in our experiment. Assadi et al. (2011) confirmed proline increase in plants growing in air polluted region. The amino acid ß-alanine accumulates markedly in response to stress in plants and it especially discussed in relation to intracellular pH regulation. According to Atanasova (2008) the increase of proline and alanine could serve as an indicator for unbalanced nitrogen nutrition. Phenylalanine and tryptophan are necessary for protein biosynthesis; phenylalanine is also a substrate for the phenylpropanoid pathway that produces numerous secondary plant products, such anthocyanins, lignin, growth promoters, growth inhibitors and phenolics. Tryptophan is the precursor for idolacetic acid, a plant hormone necessary for cell expansion. Trp plays a major role in the regulation of plant development and defense responses. Characteristically, Trp biosynthesis is induced by stresses. This finding is confirmed by our results. However, little is known about Trp-mediated trace elements tolerance (Sanjaya Hsiao et al., 2008). Sanjaya Hsiao et al. (2008) reported that increased Trp levels make Cd less accessible to the plant, decrease Cd transport and thus reduce Cd accumulation. Metal ions and the bivalent Trp side chain indole were found to interact cooperatively (Li and Yang, 2003).
5. Conclusion Results of the pot experiment confirmed the toxic effect of PM for lettuce. The effect of PM applied to soil depends on the soil characteristics, mainly on soil sorption of trace elements originating from PM and on their availability for plants. We can conclude that PM contamination of the lettuce leaves affected plant metabolism not only by trace elements from PM, but the PM toxic effect was intensified by shading of the leaf surface. The PM contamination was associated with the strong inhibition of above-ground biomass and with the enhancement of plant trace
element contents. The significant changes of amino acid levels and leaf gas-exchange parameters of the plants showed strong linear dependences on PM contamination. Limited nitrate assimilation was also confirmed. Based on our results we can confirm PM as significant damage for plant nitrogen metabolism.
Acknowledgments The presented study was supported by the research projects GA CR 521/09/1150 (Czech Science Foundation), MSM 6046070901 (Ministry of Education of the Czech Republic) and AVOZ 50380511 (Academy of Sciences of the Czech Republic).
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Trace elements present in airborne particulate matter—Stressors of plant metabolism Milan Pavlı´k a, Daniela Pavlı´kova´ b,n, Veronika Zemanova´ b, Frantiˇsek Hnilicˇka c, Veronika Urbanova´ b, Jiˇrina Sza´kova´ b a
ˇ ´ 1083, 14220 Prague, Czech Republic Isotope Laboratory, Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Vı´denska ´ 129, ´cka Department of Agro-Environmental Chemistry and Plant Nutrition, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Kamy 16521 Prague, Czech Republic c ´ 129, 16521 Prague, Czech ´cka Department of Botany and Plant Physiology, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Kamy Republic b
a r t i c l e i n f o
abstract
Article history: Received 25 August 2011 Received in revised form 12 December 2011 Accepted 13 December 2011 Available online 31 December 2011
Changes of amino acid concentrations (glutamic acid, glutamine, asparagine, aspartate, proline, tryptophan, alanine, glycine, valine and serine), gas-exchange parameters (net photosynthetic rate, transpiration rate, stomatal conductance and intercellular CO2 concentration) and nitrate levels in Lactuca serriola L. under airborne particulate matter (PM) contamination reported here reveal their role in plant chronic stress adaptation. Results of the pot experiment confirmed the toxic effect of trace elements present in PM for lettuce. PM applied to soil or on the lettuce leaves were associated with the strong inhibition of above-ground biomass and with the enhancement of plant trace element contents. The significant changes of amino acid levels and leaf gas-exchange parameters of the plants showed strong linear dependences on PM contamination (R2 ¼ 0.60–0.99). PM application on leaves intensified toxic effect of trace elements (As, Pb, Cr and Cd) originating from PM by shading of the leaf surface. The plant accumulation of nitrate nitrogen after PM contamination confirmed to block nitrate assimilation. & 2011 Elsevier Inc. All rights reserved.
Keywords: Airborne particulate matter Amino acids Gas-exchange parameters Lactuca serriola L. Toxic elements
1. Introduction Air pollution is one of the most important environmental problems in densely populated and industrialized areas. Airborne particulate matter (PM) in urban air has been at the center of recent concerns, mainly due to its adverse health effects on the urban population. Among the characteristics of particulate matter that relate to its toxicity could be mentioned the presence of trace metals (Rodriguez et al., 2010). Motor vehicles have been demonstrated to be a major contributor of particle-bound trace metals (Valavanidis et al., 2006) in urban areas. As plants are immobile and more sensitive in terms of physiological reaction to the common air pollutants than humans and animals, they better reflect local conditions. High accumulation due to airborne particulate matter was found for Pb, Cr and Cd, especially in leafy vegetables (Voutsa et al., 1996). The major contribution of most trace elements to vegetable leaves was from atmosphere. According to De Temmerman and Hoenig (2004) the leaves accumulate the deposited airborne trace elements; however, they are also influenced by soilborne metals. Mechanisms of air pollution
n
Corresponding author. Fax: þ420 234381801. E-mail address: [email protected] (D. Pavlı´kova´).
0147-6513/$ - see front matter & 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2011.12.009
toxicity are very complex and depend on various physiological and biochemical properties of plants. The PM deposited on the leaf surface can affect the plant’s metabolism by blocking light, obstructing stomatal apertures, increasing leaf temperature and altering pigment and mineral contents of the leaf (Kuki et al., 2008). Surface dust deposits may alter the optical properties of leaves, particularly the surface reflectance in the visible and short wave infrared radiation range (Hope et al., 1991). In response to these adverse effects various biochemical changes also occur such as decreased chlorophyll content and increased ascorbic acid content of leaves. The significant negative correlation between dust load and leaf pigment content in vegetation near the highway was confirmed by Prusty et al. (2003). According to Poma et al. (2002) in plant tissues an enhancement of the specific activity of the stress-related enzyme peroxidase was monitored and were confirmed genotoxic activities associated with the coarse (PM 10 smaller than 10 mm) and the fine fraction (PM 2.5 smaller than 2.5 mm) of airborne particulates. Field transect studies have shown significant negative correlations between air pollutant concentrations and net photosynthesis, biomass accumulation and yield of crop plants (Agrawal, 2005). Plant stress metabolism has been usually studied in experiments focused on short-term effects of stress agens, i.e. in the form of an acute stress. This form of stress, however, does not
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reflect correctly the environmental conditions (di Toppi and Gabbrielli, 1999). Plants usually grow and develop in conditions of long-term chronic stresses—they take up small quantities of several toxicants after a long time period. For this reason, the PM study has been focused on complex changes of plant metabolism (plant growth, photosynthesis, N metabolism, etc.) under chronic stress. Wuytack et al. (2011) recommended the use of plant characteristics, such as growth, total biomass production and specific leaf area, as diagnostic monitoring tools. Amino acid metabolism may play an important role for plant growth and development as well as in plant stress resistance, by osmotic adjustment and the accumulation of compatible osmolytes, detoxification of active oxygen species and risk elements and intracellular pH regulation (Singh, 1999). We suppose that the impact of trace elements originated from airborne particulate matter on photosynthesis and nitrogen utilization by plants can result in changes of amino acids concentrations of leafy vegetable. The present study was conducted to examine the relationship between amino acids concentrations and leaf gas-exchange parameters as a result of lettuce contamination by trace elements contained in airborne particulate matter.
Table 2 Total concentrations of selected trace elements in tested soil and in airborne particulate matter determined at the beginning of experiment. The values represent the means of data obtained in the experiment (n ¼2, i.e. two experimental years). As mg kg Chernozem Airborne particulate matter
Cd
Cr
Pb
1
39.47 0.01 0.69 7 0.07 65.5 7 2.76 30.4 7 2.2 24.57 0.85 2.06 7 0.27 125.5 7 3.27 131.5 7 8.9
was dissolved in 20 mL of 1.5% HNO3 (v/v) (electronic grade purity, Analytika Ltd., Czech Republic) and kept in glass tubes until the analysis. Aliquots of the certified reference material RM NCS DC 73350 poplar leaves (purchased from Analytika, CZ) were mineralized under the same conditions for quality assurance. Total element concentrations in PM and in soil were determined in digests obtained by two-step decomposition as follows: 0.5 g of the sample was decomposed by dry ashing in a mixture of oxidizing gases (O2 þO3 þNOx) in an Apion Dry Mode Mineralizer (Tessek, CZ) at 400 1C for 10 h; the ash was then dissolved in a mixture of HNO3 þ HF (2:1 v/v), evaporated to dryness at 160 1C and dissolved in diluted Aqua Regia (Pavlı´kova´ et al., 2008). The concentrations of trace elements were determined by ICP-OES with axial plasma configuration (Varian VistaPro, Varian, Australia). The toxic effects of As, Cd, Cr and Pb for plant was evaluated for their highest concentration in PM (Table 2).
2. Materials and methods 2.1. Plant material and cultivation conditions Adaptation of lettuce (Lactuca serriola L. var. capitata cv. Detenicka atrakce) plants to trace elements present in air pollution was investigated in pot experiment repeated for two years. For this experiment, lettuce plants (seeds obtained from SEMO Ltd. Smrzˇice, Czech Republic) were planted into plastic pots containing the soil as specified below. The plants (one plant per pot) were cultivated from April to June under natural light and temperature conditions at the experimental hall of the Czech University of Life Sciences Prague, Czech Republic. The water regime was controlled and the soil moisture was kept at 60% maximum waterholding capacity. Airborne particulate matter using for experiment was sampled in air condition units in large buildings near highway (Prague, Czech Republic) with heavy traffic (87 thousand of motor vehicles per day). We got the dust mostly from vehicular traffic with a dominance of fine particles. The fraction o 0.065 mm was used for this experiment. For cultivation of lettuce plants, 5 kg of chernozem (pHKCl ¼7.2, Cox ¼ 1.83%, CEC¼ 258 mval kg 1) was thoroughly mixed with 0.5 g N, 0.16 g P and 0.4 g K applied in the form of ammonium nitrate and potassium hydrogen phosphate for all treatments. Lettuce was exposed to a PM applied to soil (the soil was spiked prior to the experiment with 30 g PM per pot) or to the application of suspended PM on the leaves during the vegetation (Table 1). Lettuce leaves were sprayed by suspended PM eight times (3.75 g PM 100 mL 1 per pot for every spraying) during 4 weeks. The soil contents of toxic elements were analyzed as total contents and the portions of available elements are very low in this soil therefore the effect of these contents on plants are not high. Each treatment was performed in five replications. Lettuce plants were planted up to the stage of full leaves development (75 days after planting). The lettuce leaves were washed up after harvest. The plants from each pot were analyzed individually. 2.2. Analyses 2.2.1. Analyses of trace elements Plant samples were decomposed using the dry ashing procedure as follows: an aliquot ( 1 g) of the dried and powdered biomass was weighed into a borosilicate glass test-tube and decomposed in a mixture of oxidizing gases (O2 þO3 þNOx) at 400 1C for 10 h in a Dry Mode Mineralizer Apion (Tessek, Czech Republic). The ash
2.2.2. Analysis of free amino acids The amino acids from methanol þH2O extracts were determined using EZfaast amino acid analysis procedure (Phenomenex, USA). Samples were analyzed for amino acid contents by GC–MS using the Hewlett Packard 6890N/5975 MSD (Agilent Technologies, USA). Samples were separated on a ZB-AAA 10 m 0.25 mm amino acid analysis GC column under these conditions: the carrier gas (He) flow was kept constant at 1.1 ml min 1. The oven temperature program was the following: initial temperature 110 1C, a 30 1C min 1 ramp to 320 1C. The temperature of the injection port was 280 1C. A 1.5–2 ml sample was injected in split mode (1:15, v/v). MS conditions were as follows: MS source 240 1C, MS quad 180 1C, auxiliary 310 1C, electron energy was 70 eV, scan m/z range 45–450 and sampling rate was 3.5 scan s 1 (Pavlı´k et al., 2010a). The complex of free amino acids was determined (alanine, glycine, valine, leucine, isoleucine, threonine, serine, tryptophan, proline, asparagine, aspartate, methionine, glutamic acid, glutamine and lysine). The concentrations of the free glutamine, lysine and methionine were below detection limit of GC. 2.2.3. Analysis of nitrate nitrogen in plant biomass Dried above ground biomass was extracted by hot distilled water (1:10, w/v). Contents of N–NO3 were determined using segmental flow-analysis using a colorimetric method on a SKALARplus SYSTEM (Skalar, Netherlands). 2.2.4. Determination of gas-exchange parameters The net photosynthetic rate (PN), transpiration rate (E), stomatal conductance (gs) and intercellular CO2 concentration (Ci) were measured in the leaves in situ using the portable gas-exchange system LCproþ (ADC BioScientific Ltd., Hoddesdon, Great Britain) from 10:00 to 11:30 of Central European summer time the both experimental years. The irradiance was 802–821 mmol m 2 s 1 photosynthetically active radiation (PAR), the temperature in the measurement chamber was 25–26.5 1C, the CO2 concentration was 550750 cm3 m 3, the air flow rate was 205730 mmol s 1 and the duration of the measurement of each sample was 15 min after the establishment of steady-state conditions inside the measurement chamber (Hola´ et al., 2010). From these data, the water use efficiency was estimated (WUE¼ PN/E). For calculation of linear correlation (R2) Statistica for Windows version 7.0 CZ was used (StatSoft, Inc., Tulsa, OK).
3. Results Table 1 Experimental design. Treatment
Fertilization
Particulate matter o 0.065 mm (30 g per pot)
1 2 3
NPK NPK NPK
0—control Soil Plant leaves
Results of the pot experiment revealed the toxic effect of PM for lettuce plants. Plant response to the PM contamination was assessed on the basis of a decreased lettuce leaves dry matter and increased concentrations of trace elements in the above-ground biomass (Table 3). The strong inhibition of above-ground biomass was observed on the treatment 3 (PM application on lettuce leaves). Compared to the untreated control, the biomass yield of
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Table 3 Dry matter yield and concentrations of selected trace elements in lettuce (Lactuca serriola L. var. capitata cv. Detenicka atrakce) above-ground biomass. The values represent the means of data obtained in the experiment (n ¼10, i.e. two experimental years and five replications per each year). Treatment
1—Control 2—PM soil 3—PM plant
Yield of dry matter
As
Cd
g Per pot
mg kg 1 Dry matter
17.8 7 3.6 14.9 7 4.8 8.1 7 1.6
0.637 0.26 1.007 0.07 1.247 0.78
0.877 0.20 1.447 0.43 0.817 0.39
Cr
Pb
N–NO3
0.247 0.01 0.337 0.13 1.147 0.48
1.657 0.49 1.877 0.82 2.457 1.70
116 78 222 720 345 72
Table 4 Effect of PM contamination on CO2 intracellular concentration (Ci), the net photosynthetic rate (PN), the stomatal conductance (gs) and transpiration rate (E) of lettuce (75th day from sowing). From these data, the water use efficiency was estimated (WUE¼ PN/E) (n ¼30, the values represent the means of data obtained in two experimental years). Treatment
Ci (vpm)
PN (mmol CO2 m 2 s 1)
gs (mol m 2 s 1)
E (mmol H2O m 2 s 1)
WUE
1—Control 2—PM soil 3—PM plant
336.827 9.56 346.227 12.43 387.467 12.71
11.31 7 1.25 7.46 7 1.05 2.75 7 1.44
0.927 0.26 0.617 0.08 0.267 0.14
3.04 70.64 3.78 70.32 5.73 70.89
3.72 0.16 0.05
treatments 3 was reduced to ca. 45% of control treatment. PM application to soil reduced the yield only by 16% in contrast to control treatment. The highest contents of trace elements (As, Cr and Pb) were determined on treatment after PM application on leaves. Compared to the untreated control, the As content in above-ground biomass was enhanced up to 2 fold, the Pb content 1.5 fold and the Cr content 4.8 fold. Cadmium content was not increased. The Cd content in plant biomass was determined in extent 0.8170.39 mg kg 1—the highest Cd content was 1.2 mg kg 1 (the control highest content 1.07) and difference between this treatment and control treatment is not significant. Our results showed the dominant effect of As, Pb and Cr on plant metabolism from PM applied on leaves compared to Cd. Its content in above-ground biomass was significantly affected by PM application to soil and it increases more than by 65% compared to control treatment. PM application to soil affected the As, Cr and Pb contents in above-ground biomass less than PM application on leaves. The As content in above-ground biomass was enhanced up by 58%, the Cr by 38% and Pb content by 13% compared to control treatment. The effect of soil conditions (soil organic matter, cation exchange capacity and clay content) to the trace elements adsorption was significant. The behaviors of trace elements from PM were studied in two different soil samples and results confirmed the effect of soil characteristics (the results have published not yet). According many results Cd was of a greater potential of entering the human food chain than Pb. The net photosynthetic rate (PN) has been examined together with the stomatal conductance (gs), transpiration rate (E) and intercellular CO2 concentration (Ci) as potential selection marker for the detection of plant stress. Under PM contamination, the changes in leaf gas-exchange parameters—PN and gs—were similarly directed: both decreased and were lower than in control treatment (Table 4). PM application on leaves resulted in strong decrease of of PN and gs by 72% and 76%, respectively, as compared to control treatment. Stomatal conductance typically declines in response to increasing intercellular CO2 concentration. This finding was confirmed by our results—Ci was increased after PM application on leaves by 15% compared to control treatment. Transpiration rate was increased with PM contamination (Table 4). PM application on lettuce leaves showed high rate of transpiration, higher by 88% than control treatment. The close relationship between leaf gas-exchange parameters and content of trace elements in plant biomass was confirmed using linear correlation (Table 5). The contents of As, Cr and Pb showed highly significant correlation (R2 ¼0.79–0.99), whereas the
Table 5 Linear correlation (R2) between leaf gas-exchange parameters and trace elements contents in above-ground biomass of lettuce. Leaf gas-exchange parameters
As
Cd
Cr
Pb
Ci (vpm) PN (mmol CO2 m 2 s 1) E (mmol H2O m 2 s 1) gs (mol m 2 s 1) N–NO3 content (mg N kg 1 dry matter)
0.79 0.97 0.87 0.97 0.97
0.18 0.02 0.11 0.02 0.02
0.99 0.87 0.97 0.85 0.86
0.99 0.96 1.00 0.96 0.96
correlation was not found between Cd and leaf gas-exchange parameters. Changes of gas-exchange parameters confirmed the decrease of C and N assimilation as result of PM contamination (Table 4). The nitrate levels of PM treatments were raised compared to the control (Table 3). The significant correlations between leaf N–NO3 and leaf gas-exchange parameters were confirmed (R2 ¼0.90–0.99). The contents of nitrate levels and As, Cr and Pb showed significant correlation (R2 ¼0.86–0.97). Nitrogen flow through the amino acids can change dramatically in response to stress. Glutamate (Glu), glutamine (Gln), aspartate (Asp) and asparigine (Asn) are used to transfer nitrogen from source organs to sink tissues and to build up reserves during periods of nitrogen availability for subsequent use in growth, defense and reproductive processes. The changes of free amino acid concentrations after PM application are demonstrated in Fig. 1. Glu and Asp are key amino acids for biosyntheses of nucleic acids, ATP, cytokinins and chlorophyll. Concentration of these amino acids showed a downward trend after PM application. PM application on leaves resulted in strong decrease of free Glu (by 75%) and Asp (by 78%) contents compared to control treatment (Table 6). Glu together with cysteine (Cys) are important for detoxification of trace elements via formation of phytochelatins. The level of free Gln was low to non detectable in the plant samples analyzed and did not change when the plants were treated with PM. Asn levels changed drastically under stress conditions. Its significant increase was observed after PM application on soil (by 98%) and mainly on leaves (298%). The accumulation of free Asn could be associated with the remobilization of assimilated nitrogen as proteins and other substance. The concentrations of these amino acids showed strong linear correlations with As, Cr and Pb contents in plant above-ground biomass (As R2 ¼0.90–0.99; Cr R2 ¼0.62–0.94; Pb R2 ¼0.79–99) (Table 7). The Cd effect is not significant (R2 ¼0.02–0.08).
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Anthocyans,
AMP, ADP, ATP
Ornithine
Arginine
Antranilate
Glutamate-5-semialdehyde
5-aminolevulinate
Lysine Aspartate-4-semialdehyde
Aspartate
Pyrimidines
Methionine
Ascorbate-glutathione cycle
Glycine
Alanine
ne
Homocysteine al o Ox
Valine
Chlorophyll sis t he syn ot o Ph
athio Glut
Homoserine
Isoleucine
Chorismate
Phytochelatine
Threonine
Nucleic acids
4-Hydroxyphenylpyruvate
Phenylpyruvate
Hydroxyproline
Asparagine
Aminobenzoate
Lignin Proline
Purinebases
Tyrosine
Phenylalanine
Ureacycle
Glutamine,Glycine, CO 2, N 10-formyltetrahydrofolate
Tocopherols Folate
Indol
Alcaloids,
Cytokinines
Tryptophan
Coumarins,
Furanocoumarins
Polyamines
Indole auxins
Flavonoids,
Tannins
Shikimate
Damage&protection mechanismofplant against reactiveoxygenspecies
Glucose
Serine
t eta ac
Pyruvate
AcetylCoA
e
Citratecycle
lu xog 2-O
tar
at e
Cysteine Glycerophospholipids Glutamate
Threonine
Glutamine
Leucine
Fig. 1. Metabolism of amino acids.
Table 6 Content of selected free amino acids in lettuce (Lactuca serriola L. var. capitata cv. Detenicka atrakce) above-ground biomass. The values represent the means of data obtained in the experiment (n¼ 10, i.e. two experimental years and five replications per each year). Amino acid (mmol kg 1)
Glu Asp Asn Pro Ala Phe Trp Gly Val Leu Ile Thr Se a
Treatments 1—Control
2—PM soil
3—PM plant
2430 7 42 2566 7 58 35.8 7 2.8 98.4 7 11.6 302 736 52.3 7 10.6 11.3 7 2.6 33.4 7 6.7
16087 28 1178 7 32 71.07 1.9 2237 48 2947 32 33.07 9.8 20.17 5.3 30.07 7.9 37.17 14.1 27.77 3.7 24.77 5.2 1637 17 3877 21
589 7 21 571 7 19 142 7 10.9 123 7 17 264 7 36 31.3 7 4.9 29.7 7 4.9 28.8 7 5.9 51.2 7 10.9 39.4 7 4.6 28.2 7 1.7 157 7 21 361 7 19
a
34.8 7 6.1 28.7 7 4.1 a a
Concentrations of free amino acids were below detection limit of GC.
The amino acids proline (Pro) and ß-alanine (Ala) accumulates markedly in response to stress in plants. The increase of free Pro content in aboveground biomass was detected in our experiment. The most significant increase of free Pro concentration was observed after PM application to soil (by 126%) in contrast to control treatment. The increase of Pro after PM application on lettuce leaves was only by 25% compared to control treatment. The linear correlation between Pro and Cd content in plant (R2 ¼0.93) and Pro increase after PM application to soil confirmed
Table 7 Linear correlation (R2) between the contents of free amino acids and trace elements contents or N–NO3 content in above-ground biomass of lettuce. Amino acid
As
Cd
Cr
Pb
N–NO3
Glu Asp Asn Pro Ala Phe Gly Val Leu Ile Thr Ser Trp
0.97 0.99 0.90 0.09 0.81 0.90 0.98 0.98 0.08 0.06 0.83 0.80 0.98
0.02 0.02 0.08 0.93 0.16 0.13 0.03 0.03 0.90 0.96 0.20 0.23 0.01
0.87 0.63 0.94 0.06 0.99 0.40 0.58 0.60 0.55 0.10 0.31 0.28 0.84
0.96 0.79 0.99 0.01 0.99 0.58 0.75 0.76 0.46 0.02 0.48 0.45 0.95
0.99 0.93 0.98 0.02 0.92 0.78 0.91 0.92 0.18 0.01 0.69 0.66 0.99
the Cd effect mainly after root uptake. An alanine decrease after PM application was not significant (only by 13%). Its content has strong linear correlations with As, Cr and Pb contents in contrast with Pro (Table 7). Fig. 1 shows that serine (Ser) is associated with formation of glycophospholipids and glutathione via Cys biosynthesis. Free Cys concentration was below detection limit of GC. Serine and glycine (Gly) are formed as intermediate products in the photorespiratory cycle. Phosphoglycolate formed by photorespiration is the precursor for their formation, and the latter cysteine is formed. The changes of Gly were not observed. Free serine concentration of plants growing on control treatment was below detection limit of GC. The both PM treatments showed increase of Ser concentration, but difference between them was not observed.
M. Pavlı´k et al. / Ecotoxicology and Environmental Safety 79 (2012) 101–107
The importance of the branched-chain amino acids – valine (Val), leucine (Leu) and isoleucine (Ile) – as building blocks of proteins in plants is obvious. The changes of free Leu and Ile concentrations were not significant. The content of Val increased dramatically, mainly after PM application on lettuce leaves. Tryptophan (Trp) plays a major role in the regulation of plant development and defense responses. Trp is the precursor for auxin producing plant. Our results showed the significant increase of this amino acid for both PM treatments, mainly after PM application on lettuce leaves (by 163%). The inhibition of plant growth was affected by changes of free Gln, Glu, Asp and Trp concentrations. These amino acids play an importance role in syntheses of active forms of auxin and cytokinines (Fig. 1). The significant correlations between leaf N– NO3 and these amino acids were confirmed—R2 ¼0.93–0.99 (Table 7). No significant differences in results of this experiment were observed between individual experimental years.
4. Discussion Plant response to the PM toxic effect for lettuce plants was assessed on the basis of a decreased lettuce leaves dry matter, increased concentrations of trace elements and nitrates in the above-ground biomass, the changes in leaf gas-exchange parameters and amino acid concentrations. Decreased yield of dry matter and increased concentrations of trace elements in the above-ground biomass were determined for both contaminated treatments. Our data correspond with those by Pavlı´kova´ et al. (2002, 2007, 2008) who reported that excessive amounts of toxic elements in contaminated soil inhibited plant growth and development due to their phytotoxicity. Reduced growth observed at contaminated treatments may be partly ascribed to lower net photosynthetic rate; but not exclusively, since it has been argued that the reduced growth might be also due to increased tissue permeability. It might also result from inhibition of cell division (Redondo-Go´mez et al., 2011). Thus, reduction in growth can be linked to the high trace elements accumulation, as in this case plants have to spend extra energy to cope with the high trace element concentrations in the tissues (Israr et al., 2006). The strong inhibition of above-ground biomass and the significant changes in leaf gas-exchange parameters and amino acid concentrations were determined on treatment after PM application on leaves. PM application on leaves affected plants: (1) by toxic effect of trace elements from PM and (2) by shading of the leaf surface. There were very clear effects of PM contamination on PN and gs in our experiment and the decrease of PN involved a parallel decline of water use efficiency at both PM treatments of the experiment. The changes determining after PM application on leaves were increased by the shading effect. According to Hirano et al. (1995) the dust decreased the photosynthetic rate by this effect and the dust of smaller particles had a greater shading effect. Hope et al. (1991) reported that surface dust deposits may alter the optical properties of leaves, particularly the surface reflectance in the visible and short wave infrared radiation range. The parallel change of PN and gs in lettuce leaves reinforce evidence that the changes in PN could be mainly attributed to the changes in gs (Chartzoulakis et al., 2002). The decrease of gs may be related to an alteration in the K:Ca ratio in the guard cells and/or alterations in the concentration of abscisic acid, which controls stomatal movement . According to Pandey and Sharma (2003) gs reduction in plants of Brassica oleracea treated with Cr3 þ
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was presented by decreases in water potential and transpiration rates, as well as increases in diffusive resistance. The positive correlations of PN, E and gs with the stomatal length in the upper epidermis and the lower epidermis thickness, as well as the negative correlation of PN with the lamina thickness and palisade tissue thickness were observed in Zn stressed plant by Shi and Cai (2009). Stomatal limitation is often considered as an early physiological response to stress, which results in decreased PN, through limited CO2 availability in the mesophyll (Cornic, 2000). However, there is strong evidence that photosynthetic processes in the mesophyll such as RuBisCo activity, ATP supply, electron transport rate and light capture efficiency in the photosystems become impaired as the stress increases (Vaillant et al., 2005). Therefore, if the limitation of PN in plant were due to gs, there would be a reduction in intercellular CO2 concentration. The increase of Ci may be explained by modifications of RuBisCO activities of plant (Redondo-Go´mez et al., 2011). The inhibition in enzyme activity in presence of trace elements could be due to substitution of Mg2 þ in the active site of RuBisCO or chlorophyll subunits by metal ions (Siedlecka and Krupa, 2004). This substitution leads to instability of chlorophyll molecule and to decrease of photosynthesis efficiency. Trace elements have been found to decrease the Chl content and the Chl a/b ratio in many terrestrial plants (Khudsar et al., 2004). These parameters were correlated with PN. The results of Shi and Cai (2009) suggest that PN inhibition could be caused by Chl content reduction. The significantly increased Ci was determined only after PM application on leaves. PM application to soil did not affect Ci. The significant shading effect on Ci was confirmed by this finding. According to Hermle et al. (2007) the CO2 concentration in the leaf intercellular air space was increased as responses of Populus tremula to trace element dust. Gas exchange responses of plant to trace elements were attributed to leaf structural and ultrastructural changes resulting from hypersensitive-response-like processes and accelerated mesophyll cell senescence and necroses in the lower epidermis, especially along the transport pathways of trace elements in the leaf lamina. Transpiration rate was increased with PM contamination in our experiment. PM application on lettuce leaves showed high rate of transpiration, higher by 88% than control treatment. Majority of authors published decrease of E as plant response to trace elements (Hermle et al., 2007; Shi and Cai, 2009 etc.). Moreover, Hirano et al. (1995) have found that the additional absorption of incident radiation by the dust increased the leaf temperature, and consequently changed the photosynthetic rate in accordance with its response curve to leaf temperature. The increase in leaf temperature also increased the transpiration rate. Under shaded conditions, carbon uptake per unit leaf biomass is lower than under full light conditions (Van Hess and Clerkx, 2003). According to Wuytack et al. (2011) the degree of shadow needs to be taken into account in biomonitoring studies. Nitrate is a major nitrogen source for plants. The most plants devote a significant portion of their carbon and energy reserves to its uptake and assimilation. The reduction in photosynthetic rate led to a limited supply of metabolic energy and therefore to N assimilation restriction. The accumulation of nitrate nitrogen after PM application on leaves has been shown to block nitrate assimilation. Robinson et al. (1998) have found that the nitrate level was raised while nitrate reductase activity declined heavily at the polluted site of leaf during different stages of plant growth, compared to the control. Nitrate reductase is a key enzyme of nitrogen metabolism. It catalyzes the first step in the assimilatory reduction of nitrate into ammonia, which reduces nitrate to nitrite. Inhibition of nitrate reductase activity by trace element
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stress has been reported in higher plants (Nagajyoti et al., 2010). The decrease in nitrate reductase activity in trace metal treated plants could also reflect a decrease in photosynthesis because sugars are essential for nitrate reductase expression (Solanki and Dhankhar, 2011). A linear dependence of photosynthetic capacity on leaf nitrogen content was assumed by Leuning et al. (1995). PM contamination caused depletion in the pools of free glutamate and asparagines—amino acids that are metabolized during photorespiration. Glutamate, glutamine, aspartate and asparagines are used to transfer nitrogen from source organs to sink tissues and to build up reserves during periods of nitrogen availability for subsequent use in growth, defense and reproductive processes. These compounds are the major amino acids translocated in the phloem of most species. Gln is not only the major amino acid used for nitrogen transport, but also a key metabolite that acts as an amino donor to other free amino acids, primarily catalyzed by glutamate synthase. This pathway interacts with carbohydrate metabolism or the energy status of the organ (Hodges et al., 2002). Asparagine accumulates under conditions of stress. There is evidence that asparagine can bind to cadmium, lead and zinc. Cadmium stress on Zea mays L. decreased the glutamine and asparagine content. The findings of Pavlı´k et al. (2010b) have confirmed these results and have showed significant decrease of asparagine concentration after arsenic stress on spinach. Lea et al. (2007) have published opposite results. Cadmium induced an almost 10-fold increase in the asparagine concentration of tomato roots but only fourfold in the leaves. Accumulation of free proline in plants has often been reported as a consequence of a wide range of environmental stress (Pavlı´kova´ et al., 2008). The increase of free proline content in aboveground biomass was detected in our experiment. Assadi et al. (2011) confirmed proline increase in plants growing in air polluted region. The amino acid ß-alanine accumulates markedly in response to stress in plants and it especially discussed in relation to intracellular pH regulation. According to Atanasova (2008) the increase of proline and alanine could serve as an indicator for unbalanced nitrogen nutrition. Phenylalanine and tryptophan are necessary for protein biosynthesis; phenylalanine is also a substrate for the phenylpropanoid pathway that produces numerous secondary plant products, such anthocyanins, lignin, growth promoters, growth inhibitors and phenolics. Tryptophan is the precursor for idolacetic acid, a plant hormone necessary for cell expansion. Trp plays a major role in the regulation of plant development and defense responses. Characteristically, Trp biosynthesis is induced by stresses. This finding is confirmed by our results. However, little is known about Trp-mediated trace elements tolerance (Sanjaya Hsiao et al., 2008). Sanjaya Hsiao et al. (2008) reported that increased Trp levels make Cd less accessible to the plant, decrease Cd transport and thus reduce Cd accumulation. Metal ions and the bivalent Trp side chain indole were found to interact cooperatively (Li and Yang, 2003).
5. Conclusion Results of the pot experiment confirmed the toxic effect of PM for lettuce. The effect of PM applied to soil depends on the soil characteristics, mainly on soil sorption of trace elements originating from PM and on their availability for plants. We can conclude that PM contamination of the lettuce leaves affected plant metabolism not only by trace elements from PM, but the PM toxic effect was intensified by shading of the leaf surface. The PM contamination was associated with the strong inhibition of above-ground biomass and with the enhancement of plant trace
element contents. The significant changes of amino acid levels and leaf gas-exchange parameters of the plants showed strong linear dependences on PM contamination. Limited nitrate assimilation was also confirmed. Based on our results we can confirm PM as significant damage for plant nitrogen metabolism.
Acknowledgments The presented study was supported by the research projects GA CR 521/09/1150 (Czech Science Foundation), MSM 6046070901 (Ministry of Education of the Czech Republic) and AVOZ 50380511 (Academy of Sciences of the Czech Republic).
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