Sunflower Plants as Bioindicators of Environmental Pollution with Lead (II) Ions

Sensors 2009, 9, 5040-5058; doi:10.3390/s90705040 sensors ISSN 1424-8220 www.mdpi.com/journal/sensors Article Sunflower Plants as Bioindicators of Environmental Pollution with Lead (II) Ions Olga Krystofova 1, Violetta Shestivska 1,2, Michaela Galiova 3, Karel Novotny 3, Jozef Kaiser 4, Josef Zehnalek 1, Petr Babula 5, Radka Opatrilova 5, Vojtech Adam 1,6 and Rene Kizek 1,* 1 Department of Chemistry and Biochemistry, Mendel University of Agriculture and Forestry, Zemedelska 1, CZ-613 00 Brno, Czech Republic 2 Department of Plant Biology, Mendel University of Agriculture and Forestry, Zemedelska 1, CZ-613 00 Brno, Czech Republic 3 Department of Chemistry, Faculty of Science, Masaryk University, Kotlarska 2, CZ-611 37 Brno, Czech Republic 4 Institute of Physical Engineering, Faculty of Mechanical Engineering, Brno University of Technology, Technicka 2896/2, CZ-616 69 Brno, Czech Republic 5 Department of Natural Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Palackeho 1-3, CZ-612 42 Brno, Czech Republic 6 Department of Animal Nutrition and Forage Production, Faculty of Agronomy, Mendel University of Agriculture and Forestry, Zemedelska 1, CZ-613 00 Brno, Czech Republic * Author to whom correspondence should be addressed; E-Mail: [email protected]uni.cz Received: 31 May 2009; in revised form: 22 June 2009 / Accepted: 24 June 2009 / Published: 25 June 2009 Abstract: In this study, the influence of lead (II) ions on sunflower growth and biochemistry was investigated from various points of view. Sunflower plants were treated with 0, 10, 50, 100 and/or 500 µM Pb-EDTA for eight days. We observed alterations in growth in all experimental groups compared with non-treated control plants. Further we determined total content of proteins by a Bradford protein assay. By the eighth day of the experiment, total protein contents in all treated plants were much lower compared to control. Particularly noticeable was the loss of approx. 8 µg/mL or 15 µg/mL in shoots or roots of plants treated with 100 mM Pb-EDTA. We also focused our attention on the activity of alanine transaminase (ALT), aspartate transaminase (AST) and urease. Activity of the enzymes increased with increasing length of the treatment and applied concentration OPEN ACCESS Sensors 2009, 9 5041 of lead (II) ions. This increase corresponds well with a higher metabolic activity of treated plants. Contents of cysteine, reduced glutathione (GSH), oxidized glutathione (GSSG) and phytochelatin 2 (PC2) were determined by high performance liquid chromatography with electrochemical detection. Cysteine content declined in roots of plants with the increasing time of treatment of plants with Pb-EDTA and the concentration of toxic substance. Moreover, we observed ten times higher content of cysteine in roots in comparison with shoots. The observed reduction of cysteine content probably relates with its utilization for biosynthesis of GSH and phytochelatins, because the content of GSH and PC2 was similar in roots and shoots and increased with increased treatment time and concentration of PbEDTA. Moreover, we observed oxidative stress caused by Pb-EDTA in roots where the GSSG/GSH ratio was about 0.66. In shoots, the oxidative stress was less distinctive, with a GSSG/GSH ratio 0.14. We also estimated the rate of phytochelatin biosynthesis from the slope of linear equations plotted with data measured in the particular experimental group. The highest rate was detected in roots treated with 100 µM of Pb-EDTA. To determine heavy metal ions many analytical instruments can be used, however, most of them are only able to quantify total content of the metals. This problem can be overcome using laser induced breakdown spectroscopy, because it is able to provide a high spatial-distribution of metal ions in different types of materials, including plant tissues. Data obtained were used to assemble 3D maps of Pb and Mg distribution. Distribution of these elements is concentrated around main vascular bundle of leaf, which means around midrib. Keywords: phytoremediation; heavy metals; sunflower; lead ions; high performance liquid chromatography with electrochemical detection; spectrometry; laser induced breakdown spectroscopy 1. Introduction Environmental remediation deals with the removal of contaminants from soil, groundwater, sediment, surface water etc. for the general protection of human health and the environment [1]. Remediation processes can be expensive, as the are mostly ex-situ methods involving excavation of impacted soils and subsequent treatment at the surface. Therefore new, efficient, inexpensive and nonenvironmentally disruptive technologies are still developing. One of the groups of such new technologies is called bioremediation. It involves the treatment of environmental problems through organisms. Microorganisms (e.g., Desulfomonile, Clostridium, Pseudomonas, Acinetobacter) and plants (e.g., Betula, Populus) are most commonly used for these purposes [2-7]. If plants are used, we call this process phytoremediation [1,8-10]. A range of processes mediated by plants are useful in treating environmental problems. Plants can chemically modify toxic substances as a direct result of plant metabolism (phytotransformation), can reduce the mobility of substances in the environment (phytostabilization) or uptake and concentrate substances from the environment into the plant biomass (phytoextraction). The scheme of various ways how to a plant metabolizes or deposit the pollutant is shown in Figure 1. Sensors 2009, 9 5042 Figure 1. Phytoremediation can occur through a series of complex interactions between plants, microbes, and the soil, including accumulation, hyperaccumulation, exclusion, volatilization, and degradation of the target pollutant. Plants also stabilize mobile contaminated sediments by forming dense root mats inside soil. Volatilization Transpiration Inorganics Organics Accumulation Heavy metals, Radionuclides, Metabolites Rhizosphaeric metabolism Metals, Organics, Radionuclides Heavy metals, Radionuclides Chlorinated hydrocarbons Mercury, Selenium Volatilization Transpiration Inorganics Organics Accumulation Heavy metals, Radionuclides, Metabolites Heavy metals, Radionuclides, Metabolites Rhizosphaeric metabolism Metals, Organics, Radionuclides Heavy metals, Radionuclides Chlorinated hydrocarbons Mercury, Selenium Most toxic substances (organic pollutants, heavy metals) come from anthropogenic activities such as mining, traffic, heavy industry, etc. [11-13]. Contrary to organic pollutants, heavy metals cannot be degraded or destroyed. To a small extent they enter our bodies via food, drinking water and air. As trace elements, some heavy metals (e.g., copper, selenium, zinc) are essential to maintain the metabolism of the human body. However, others such as cadmium, lead, and mercury are toxic at all. At higher concentrations both groups of heavy metals (toxic and essential) can lead to poisoning [13]. Heavy metals are also dangerous because they tend to bioaccumulate. Lead is one of the most dangerous and toxic heavy metals. Levels of lead in the environment are not stable and vary according to industrial production, urbanization, climate changes and many other factors [14]. The levels of lead in the environment vary between 4 and 20 mg/g of dust. Uncontaminated waters contain lead in concentrations ranging from 0.001 to 0.06 mg/L. In soils, levels of lead reach 5 to 30 mg per kg of soil. When lead is added into petrol as an additive, the highest lead levels are determined on the surfaces of leaves, from where lead enters the food chain, as well as soil or water. Lead is present in soils as salts in soluble as well as insoluble forms. Lead contamination in the soil is known to inhibit seed germination [15,16]. The inhibition of germination by exogenously supplied Pb2+ is a possible effect of interference with some important enzymes involved in the process. Photosynthesis is considered as one of the metabolic processes most sensitive to Pb2+ toxicity [17]. Closing of the stomata, disruption of the chloroplastic organization, change in the metabolites of photosynthesis and replacement of essential ions like magnesium are the main effects on photosynthesis of lead toxicity [14,18-21]. The Sensors 2009, 9 5043 metal has also been reported to inhibit photosynthesis in isolated chloroplasts. There have been also published data reporting on inhibition of enzymes crucial for nitrogen assimilation [14]. Figure 2. Experimental arrangement of LIBS: 1 – Nd:YAG laser, 2 – modulator of second harmonic frequency, 3 – periscope, 4 – CCD camera, 5 – ablation chamber, 6 – fibre optic system, 7 – monochromator, 8 – ICCD camera. As we mentioned above for the particular example of lead ions, there are many mechanisms and pathways which can be affected by heavy metals [22-25]. Protective mechanisms of a plant cell against the toxic effects of heavy metals mainly involve synthesis of compounds rich in cysteine called phytochelatins. Their synthesis comes from the most abundant thiol – reduced glutathione. To detect these compounds many various methods and techniques have been employed [23,24,26-30], including sensors and biosensors [31-33]. However, uptake and transport as well as storage of heavy metals through plant tissues remain still unclear. To consider whether a specific plant species is able or not able to remediate the polluted environment, not only heavy metals content in the plant tissues, but also the distribution of such metal ions in the tissues must be analysed. Analytical methods and instruments for detection of lead (II) ions have been reviewed several times [34-38]. The diagnostic techniques enabling monitoring high spatialand lateral-distribution of elements within different plant structures include mainly X-ray imaging methods [39-41]. X-ray microscopy and micro-radiography investigations usually make use of soft X-rays generated by plasma laser, microfocus X-ray sources and synchrotron radiation [42]. Although the X-ray radiation based methods are relatively high-cost and availability of the experimental apparatus is limited due to possibility of “in-situ” analysis only, it offers new aspects for studying the distribution of heavy metals. However, X-ray imaging methods are intensively investigated in our laboratories; recently we have been focusing also on the realization of spatially-resolved spectro-chemical analysis by utilizing laser-ablation based techniques. Laser induced breakdown spectroscopy (LIBS, Figure 2) is a type of atomic emission spectroscopy which utilises a highly energetic laser pulse as the excitation source and is able to provide high spatialdistribution of metal ions in different types of materials [43-45]. The character of the ablative process Sensors 2009, 9 5044 depends on the features of the laser used (wavelength, pulse duration, power and energetic profile of the rays), surrounding atmosphere and the features of the sample itself (matrix, absorption characteristics, its structure) [46]. LIBS method is one of analytical instruments which makes qualitative and quantitative analysis and also monitoring of element distribution in different types of samples possible. The main advantage of this method is that it requires no, or minimal sample pretreatment and enables multi-elemental analysis with high three-dimensional resolution. A limiting factor is especially the diameter of laser ray. In this study, the influence of lead (II) ions on sunflower plants (Figure 3) was investigated from various points of view. We aimed our study at common growth parameters, morphological changes, total protein content, activity of certain enzymes, level of stress induced thiols and spatial distribution of lead. Figure 3. Pictures of sunflower plants in the second, sixth and eighth experimental day after Pb-EDTA application (0, 10, 50, 100 and 500 µM). 2nd day 0 µM 10 µM 50 µM 100 µM 500 µM 6th day 8th day 10 cm Sensors 2009, 9 5045 2. Results and Discussion 2.1. Morphological changes Lead is a poisonous metal that has many adverse effects on plants and animals. Sunflower plants were treated with 0, 10, 50, 100 and/or 500 µM Pb-EDTA for eight days. We observed alterations in growth in all experimental groups compared with non-treated control plants. Plants exposed to lead (II) ions grew faster in comparison with control plants, except for the highest applied concentration. This phenomenon probably relates to the stimulatory effects of the presence of Pb-EDTA, because control plants were cultivated in distilled water only, where no other nutrients are present. In addition we observed chlorosis on plants treated with the highest concentration (Figure 3). When we compared the fresh weight of plants treated with lead (II) ions with the non-treated experimental group, it was possible to clearly notice the effect of applied Pb-EDTA concentration on the aerial parts of plants, except for the highest applied concentration (Figure 4 A). Figure 4. Changes of (A) fresh and (B) dry weights of sunflower plants exposed to PbEDTA. Dry mass was obtained by drying to the constant weight at 105 °C in the oven. All data were obtained by subtraction from control plants. The experiment was carried in triplicates. AB Length of treatment (d) Length of treatment (d) -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 2468 -0.025 -0.020 -0.015 -0.010 -0.005 0.000 0.005 0.010 2468 0.0 0.1 0.2 0.3 0.4 10 µM Pb 2+ 50 µM Pb 2+ 100µM Pb 2+ 500µM Pb 2+ 10 µM Pb 2+ 50 µM Pb 2+ 100µM Pb 2+ 500µM Pb 2+ Shoot Root -0.10 -0.08 -0.06 -0.04 -0.02 0.00 0.02 0.04 Dry weight (g) Fresh weight (g) Shoot Root 10 µM Pb 2+ 50 µM Pb 2+ 100µM Pb 2+ 500µM Pb 2+ 10 µM Pb 2+ 50 µM Pb 2+ 100µM Pb 2+ 500µM Pb 2+ Sensors 2009, 9 5046 Nevertheless, the adverse effect of lead (II) ions is shown on dependence of dry weight on length of the treatment and applied concentration (Figure 3B). Determined change is probably connected with increased water uptake of plants exposed to stress caused by heavy metals. This hypothesis is supported by results reporting on nuclear magnetic resonance analysis of early somatic embryos clusters [29]. In the case of change in fresh weight of roots, increases by the sixth and eighth day were detected, except for the highest applied concentration. Dry weight of roots decreased, except on the second day of the treatment for all experimental groups (Figure 4 A,B). 2.2. Total protein content Heavy metals taken up by plants or induce stress reactions, which manifest as enhancements of the levels of certain molecules. Firstly, the level of mRNA is enhanced with subsequent changes in protein profile. Therefore, we determined total content of proteins by a Bradford protein assay. Total content of proteins slightly increased in both aerial parts and roots in the second day. From the fourth day of the treatment, a decrease of total content of proteins occurred. In eighth day of the experiment this loss was approx. 8 µg/mL or 15 µg/mL in shoots or roots of plants treated with 100 mM Pb-EDTA (Figure 5). Total content of proteins is dramatically reduced thanks to the heavy metal ions. This trend matches well with the dry weight dependence (Figures 4 and 5). Figure 5. Changes in total protein content in sunflower plants exposed to Pb-EDTA. All data were obtained by subtraction from control plants. The experiments were carried out in triplicate. Length of treatment (d) -20 -15 -10 -5 0 5 10 2468 -10 -8 -6 -4 -2 0 2 Total protein content (mg/ml) Shoot Root 10 µM Pb 2+ 50 µM Pb 2+ 100µM Pb 2+ 500µM Pb 2+ 10 µM Pb 2+ 50 µM Pb 2+ 100µM Pb 2+ 500µM Pb 2+ Sensors 2009, 9 5047 2.3. Determination of plant enzymes’ activity There is still not much available information about the significance of some commonly analyzed enzymes as markers of stress reactions in plants. In several papers, we have demonstrated that some enzymes (such as aminotransferases or urease) can participate in plant stress reactions [22,26,47-51]. Thus, we focused our attention on the activity of alanine transaminase (ALT), aspartate transaminase (AST) and urease. Transaminases catalyze the transfer of the amino groups of amino acids to 2-oxoacids. In plants, transaminases participate very effectively in transformations of nitrogen compounds. They are important for the synthesis of amino acids from oxo-acids in the citrate cycle and for other crucial biochemical pathways. They also play key roles in the synthesis of secondary metabolites as well as chlorophyll. In roots AST and ALT activities were increased during the experiments in comparison to control plants (Figure 6). This increase corresponds well with the higher metabolic activity. Urease activity was enhanced in aerial plant parts as well as in roots with increasing length of exposition and applied concentration slightly (data not shown). Figure 6. Changes of AST and ALT activities in sunflower plants exposed to Pb-EDTA. All data were obtained by subtraction from control plants. The experiment was carried in triplicates. Length of treatment (d) Length of treatment (d) -0.8 -0.6 -0.4 -0.2 0 0 1 2 3 4 5 2 4 6 8 -10 -8 -6 -4 -2 0 AST Shoot -4 0 4 8 12 16 2468 Root Catalytic activity (µkat/l) Shoot Root ALT Catalytic activity (µkat/l) 10 µM Pb 2+ 50 µM Pb 2+ 100µM Pb 2+ 500µM Pb 2+ 10 µM Pb 2+ 50 µM Pb 2+ 100µM Pb 2+ 500µM Pb 2+ 10 µM Pb 2+ 50 µM Pb 2+ 100µM Pb 2+ 500µM Pb 2+ 10 µM Pb 2+ 50 µM Pb 2+ 100µM Pb 2+ 500µM Pb 2+ 2.4. Content of low molecular mass thiols Low molecular mass compounds rich in cysteine moieties play a very important role in the ability to withstand or even hyperaccumulate heavy metals ions. Due to this, we paid attention to such compounds. Particularly, the contents of cysteine, reduced glutathione (GSH), oxidized glutathione Sensors 2009, 9 5048 (GSSG) and PC2 were determined by high performance liquid chromatography with electrochemical detection (Figure 7). Contents of cysteine differed markedly in shoots and roots. Cysteine content declined in the roots of plants as the time of the treatment of plants with Pb-EDTA and concentration of toxic substance increased. Moreover, we observed ten times higher content of cysteine in roots in comparison with shoots. The observed reduction of cysteine content probably relates to its utilization for the biosynthesis of GSH and phytochelatins. Content of GSH was similar in roots and shoots and increased with increasing time of the treatment and concentration of Pb-EDTA (Figure 7). We plotted the dependence with linear regression to estimate the rate of synthesis of GSH. The rate expressed as the slope of the linear equation was 0.531x and 0.635x for roots and shoots, respectively, where “x” is concentration of applied Pb-EDTA. Figure 7. Changes of cysteine, GSH, GSSG and PC2 contents in sunflower plants exposed to Pb-EDTA. All data were obtained by subtraction from control plants. The experiment was carried out in triplicate. Cys Cysteine content (µg/g) shoot root GSH GSSG PC2 Reduced glutathione content (µg/g) Phytochelatin2 content (µg/g) 2 4 6 8 2 4 6 8 2 4 6 8 2 4 6 8 2 4 6 8 2 4 6 8 2 4 6 8 2 4 6 8 Length of treatment (d) Length of treatment (d) Length of treatment (d) Length of treatment (d) Oxidized glutathione content (µg/g) 10 µM Pb 2+ 50 µM Pb 2+ 100µM Pb 2+ 500µM Pb 2+ 10 µM Pb 2+ 50 µM Pb 2+ 100µM Pb 2+ 500µM Pb 2+ It is a common knowledge that heavy metals induce generation of free oxygen species, which subsequently damage cell compartments (membranes, nucleic acids). Oxygen radicals can be scavenged by various mechanisms inside a cell [52]. Low molecular mass thiols are able to react with oxygen radicals via formation of disulphides. One of the most studied and well known reactions of such type is the redox cycling of GSH into GSSG [53]. In our experiment, GSSG levels gradually increased (Figure 7). 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