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).

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).

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).

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-oxo-acids. 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).

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 (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.

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). We observed oxidative stress caused by Pb-EDTA in roots when the GSSG/GSH ratio was about 0.66. In shoots, the oxidative stress was less distinctive, with a GSSG/GSH ratio of 0.14. It follows from the results obtained that only a small part of the up-taken lead(II) ions is transported into shoots (stems, leaves). The content of PC2 in roots and shoots is shown in Figure 7. With increasing time of the treatment and concentration of Pb-EDTA the content was markedly enhanced. Moreover, the ability of plants to withstand oxidative stress caused by heavy metal depends also on rate of phytochelatin biosynthesis. Therefore, we again estimated the rate of phytochelatin biosynthesis via the slope of the linear equations plotted with data measured in each particular experimental group. The highest rate was detected in roots treated with 100 μM of Pb-EDTA (11.200) followed by 50 μM (13.270), 500 μM (7.100) and 10 μM (6.500). Compared to roots, the rate of phytochelatin biosynthesis was lower in shoots. The highest rate was detected in shoots treated with 50 μM of Pb-EDTA (4.600) followed by 100 μM (4.500), 500 μM (4.300) and 10 μM (2.719). It can be concluded that protective metabolic pathways of plants treated with 50 and 100 μM of Pb-EDTA was stimulated to withstand the adverse effect of heavy metal ions.

To determine heavy metal ions many analytical instruments can be used, however, most of them are only able to quantify total contents of the metals [38,54–60]. This problem can be overcome using LIBS, because it is able to provide high spatial-distribution of metal ions in different types of materials [19,20,22,42,61,62]. Recently, we published the first experimental data focused on utilization of the LIBS technique for analysis of biological samples exposed to various heavy metals. It was demonstrated that the mentioned techniques are a unique analytical tool that can provide biologically interesting data [61,62]. The laser-generated patterns consisting of precisely ablated micro-craters have been utilized for mapping the lead and magnesium distribution on 4.5 × 2 mm² leaf sections of sunflower samples. Measurement of optical emission of atoms by ICCD camera was carried out under optimized conditions. A typical LIBS spectrum after application of one laser pulse is shown in Figure 8.

Pb and Mg lines were identified in the measured spectral range. The analytical line of Pb (I) at 283.31 nm was used for detection of Pb (I). From the emission lines of magnesium, the 277.98 nm Mg (I) analytical line was selected. It was not possible to use other magnesium emission lines due to detector saturation. Subtraction of background was realized and the area under the emission line was calculated for all measurements.

The data were used to assemble 3D Pb and Mg distribution maps. The Pb and Mg distribution in samples of maize leaf was relatively homogenous (Figure 9A). The three-dimensional distribution of Pb and Mg obtained by ablation of sunflower leaf samples exposed to 0.5 mM Pb-EDTA and the ablative patterns are shown in Figure 9B. The midrib is clearly evident in the sample due to distribution of Pb in this area. The distribution of Mg ions in this sample is homogenous, like in maize leaves. In the case of leaf samples of lettuce plant exposed to 0.5 mM Pb-EDTA, the highest content of both elements was determined in the midrib. This fact is clearly shown in Figure 9C. We also investigated the distribution of Pb and Mg in leaf samples of the same plants treated with 1 mM Pb-EDTA (3D maps on the right side in Figure 9). In these samples, we can see a heterogeneous distribution of Mg ions as well as Pb ions. Distribution of these elements is concentrated around the main vascular bundle of leaf, which means around the midrib. In all samples, LIBS measurements were compared with those obtained by laser ablation inductively coupled plasma mass spectrometry [41,42,63]. The results obtained were in good agreement. In the view using LIBS for monitoring of element accumulation in plant materials, the instrumentation is supplemented by software, which enables fully automated measurement [46].

Acetonitrile and methanol (HPLC purity) were purchased from Merck (Darmstadt, Germany). Urease EC 3.5.1.5 (Jack Beans, type III; 45,000 IU/g) was purchased from Sigma Aldrich (St. Louis, MI, USA). Standards PC₂, PC₅ and DesGlyPC with purity higher than 90% were synthesized at Clonestar Biotech (Brno, Czech Republic). All other used chemical were also purchased from Sigma Aldrich, unless noted otherwise. The standard stock solutions (100 μg/mL) were prepared in ACS water (ie, chemicals that meet the specifications of the American Chemical Society) and stored in dark at 4 °C.

Sunflower plants (Helianthus annuus L., Compositae) were used in our experiments. Sunflower kernels were germinated in the dark on wet filter paper in special vessels at 23 ± 2 °C. After ten days, maize seedlings were placed into vessels containing distilled water and cultivated in a Versatile Environmental Test Chamber (MLR-350 H, Sanyo, Japan) for eight days with 14 h daylight per day (maximal light intensity was about 100 μE/m²s¹) at a temperature 23.5–25 °C and humidity 71–78%. After that, Pb-EDTA was added to the cultivation solution at final concentrations of 0, 10, 50, 100 and 500 μM. Plants grown without Pb-EDTA were used as a control. The sunflower plants placed in the vessels that distilled water with addition of Pb-EDTA (0, 10, 50, 100 and 500 μM) were grown for six days. Four plants each were harvested at certain time intervals (2^nd, 4^th, 6^th and 8^th day of the experiments), and their roots were rinsed three times in distilled water and 0.5 M EDTA. In addition, each harvested plant was divided into shoots (aerial plant parts) and roots. Fresh weight of the samples was measured immediately after the rinsing by using a Sartorius scale.

Weighed plant tissues (approximately 0.2 g) were transferred to a test-tube, and liquid nitrogen was added. The samples were frozen to disrupt the cells [48]. The frozen sample was transferred to mortar and ground for 1 min. Then, 1,000 μL of 0.2 M phosphate buffer (pH 7.2) was added to the mortar, and the sample was grinding for 5 min. The homogenate was transferred to a new test-tube. The mixture was homogenised by shaking on a Vortex–2 Genie (Scientific Industries, New York, USA) at 4 °C for 30 min. The homogenate was centrifuged (14,000 g) for 30 min at 4 °C using a Universal 32 R centrifuge (Hettich-Zentrifugen GmbH, Tuttlingen, Germany). Before the analysis the supernatant was filtered through a membrane filter (0.45 μm Nylon filter disk, Millipore, Billerica, MA, USA).

The HPLC-ED system consisted of two solvent delivery pumps operating in the range of 0.001–9.999 mL·min⁻¹ (Model 582 ESA Inc., Chelmsford, MA), a Metachem Polaris C18A reverse-phase column (150 × 4.6; 3 μm particle size, Varian Inc., CA, USA) and a CoulArray electrochemical detector (Model 5600A, ESA). The electrochemical detector includes three flow cells (Model 6210, ESA, USA). Each cell consists of four analytical cells. One cell contains the working carbon porous electrode, two auxiliary and two reference electrodes. Both the detector and the reaction coil/column were thermostatted. The sample (10 μL) was injected using autosampler (Model 540 Microtiter HPLC, ESA, USA). For other experimental conditions see [24,28].

Spectrometric measurements were carried using an automated chemical analyser BS-200 (Mindray, China). Reagents and samples were placed on cooled sample holder (4 °C) and automatically pipetted directly into plastic cuvettes. Incubation proceeded at 37 °C. The mixture was consequently stirred. The washing steps with distilled water (18 mΩ) were done in the midst of the pipetting. The apparatus was operated using the BS-200 software (Mindray, China).

To realize the measurements with high-spatial resolution, the sample holder with the investigated species was placed to the stage with precision movements (2 μm in x, y and z direction) inside the ablation chamber (Tescan, Czech Republic). The single-shot LIBS analysis was performed in air under atmospheric pressure. The ablation spot was targeted and controlled for each shot by a CCD camera placed outside of the chamber. The LIBS micro-plasma was created using the second harmonic (532 nm) of a Nd:YAG laser system (Quantel, Brilliant B). The laser pulse width was ∼5 ns and the beam diameter 8 mm. The energy of the laser pulse was 10 mJ (at the sample). The laser-induced plasma was produced by focusing the laser beam with a 30 mm focal-length glass doublet (Sill Optics). Imaging system consisting of two quartz objectives was used to collect the LIBS micro-plasma radiation. Subsequently, the radiation was transported by a 3 m fibre optic system onto the entrance slit of the 0.32 m monochromator (Jobin Yvon TRIAX 320). In this study the grating 2,400 g/mm of the monochromator and 50 μm entrance slit were used. The dispersed spectrum of the plasma radiation was detected by an ICCD camera (Jobin Yvon Horiba). The time-resolved measurements were realized triggering the camera by the Q-switch signal of the laser. The detector was gated 1 μs after the Q-switch signal and the observation window was 10 μs. The lead-content within the leaf was detected by monitoring the 283.31 nm Pb (I) line in the created micro-plasmas.