Fig 1.
Experimental design and methods used in individual experimental variants.
Lemna trisulca plants incubated in darkness for 24 h in control medium (a), with 15 μM Pb2+ (b), with 10–3M H2O2 added for the last 3 h of incubation (c), with 100 U catalase (d), or with Pb and catalase simultaneously (e); plants pre-incubated in darkness for 24 h and then exposed to white light for 1 h (weak 50 μE m-2s-1 or strong 1000 μEm-2s-1) in control medium (f-g) or with Pb (h-i); plants pre-incubated in darkness for 24 h and then exposed to blue light (for 45 min weak 2 μE m-2s-1 and next for 45 min strong 113 μE m-2s-1) in control medium (j-k) or with Pb (l-m).
Fig 2.
Distribution of chloroplasts in mesophyll cells of Lemna trisulca fronds in darkness.
In control plants (A, B) and in plants treated with lead (15 μM Pb2+; C-F), hydrogen peroxide (10–3 M H2O2; G, H), catalase (100 U; I, J) or simultaneously with lead and catalase (15 μM; K, L) for 24 h (in the case of H2O2, plants were treated with it for the last 3 h only). The larger micrographs show 3D reconstructions of frond fragments, obtained on the basis of a series of optical sections adjacent to the surface of fronds in a confocal microscope (S1 Fig.). The reconstructions include chloroplasts from a single layer of mesophyll cells (in some places tiny epidermal chloroplasts are also visible). A color scale is used to highlight the image depth (warm colors represent chloroplasts located closer to the viewer, while the cool ones, the more distant organelles). The smaller micrographs show chloroplast distribution (red autofluorescence) in cell cross-sections in single optical sections (S1 Fig.). The white rectangle presents a schematic outline of a cross-section of a single cell. The white arrows indicate chloroplast-free areas visible in 3D reconstruction and the corresponding locations in cross-sections. Blue arrows (E, F) indicate places in which chloroplast clusters are present, while the white arrowhead (G) points to the area neighboring the anticlinal wall, where chloroplasts were absent. Scale bar = 20 μm.
Fig 3.
Percentage of chloroplasts adopting the face or profile position per mesophyll cell of Lemna trisulca in various experimental variants.
During incubation, control plants and plants treated with lead (15 μM Pb2+) were placed in darkness for 24 h, weak white light (WW; 50 μE m-2s-1, 1 h after 24 h in darkness or strong white light (SW; 1000 μEm-2s-1, 1 h after 24 h in darkness). In two variants, plants were treated with catalase (100 U, CAT) alone or with Pb in darkness for 24 h. For comparison with the reaction to lead, some plants were treated with H2O2 alone (10–3 M) for the last 3 h in darkness. Different small letters (a-g) denote statistically significant differences (p < 0.05).
Fig 4.
Summary of results shown in Figs. 2–5.
A diagram of chloroplast distribution in cross-sections of Lemna trisulca cells of control plants and plants treated with lead, hydrogen peroxide (H2O2), catalase (CAT), or simultaneously with lead and catalase in darkness (D) and weak or strong white light (WW or SW, respectively). The numbers show the percentage of chloroplasts in the profile position in the particular variants (in ascending order). Two numbers under a cell indicate an absence of significant differences between two experimental variants. The symbol ≤ denotes that differences between Pb-SW and both Pb-D and H2O2-D are not significant but they are significant between control-SW and both Pb-D and H2O2-D. Also control-D and Pb-WW did not differ in the number of chloroplasts in the profile position, but the variants were illustrated separately in view of a difference in chloroplast distribution (marked with white arrowheads). The diagram shows three basic chloroplast positions in control variants: chloroplast accumulation (in WW light), dark position (in D), and chloroplast avoidance (in SW).
Fig 5.
Distribution of chloroplasts in mesophyll cells of Lemna trisulca in white light.
Control plants (A, B, E, F, I, J) and plants treated with lead (15 μM Pb2+; C, D, G, H, K, L) for 24 h in darkness (A-D), followed 1 h by weak white light (E-H; 50 μE m-2s-1) or strong white light (I-L; 1000 μEm-2s-1). Other explanations as in Fig. 2 and S1 Fig. Scale bar = 20 μm.
Fig 6.
Chloroplast displacements in mesophyll cells of Lemna trisulca in blue light.
Weak blue (WB, 2 μE m-2s-1) and strong blue light (SB, 113 μE m-2s-1) in the presence of lead (15 μM Pb2+) and in control plants. The line graph (A) illustrates changes in the location of chloroplasts (recorded on the basis of red light transmittance) during 45 min of exposure to constant weak blue light (a thin arrow marks when the light was turned on; darkness was the starting position) and during 45 min of exposure to strong blue light (a thick arrow marks when the strong light was turned on). The column diagrams (B-D) show amplitude of accumulation [ΔT(+), marked as T(+)] and avoidance [ΔT(-), marked as T(-)] as well as the maximum amplitude [ΔTmax = ΔT(-) + ΔT(+), marked as Tmax]; absolute velocity (% min-1) of accumulation [V(+)] and avoidance [V(-)]; and relative velocity (% min-1/ΔTmax) of accumulation [V(+)] and avoidance [V(-)]. **p < 0.05.
Fig 7.
Visualization of actin filaments Lemna trisulca mesophyll cells.
With the help of Alexa Fluor 488—phalloidin (green fluorescence) in control plants (A-C) and plants treated with lead (D-G) in darkness. Chloroplasts are visible thanks to the red autofluorescence of chlorophyll. The network of microfilament bundles varying in thickness, which were not directly connected with chloroplasts, is marked with double arrows. Microfilament bundles twisting around plastids (single white arrows), with their branches, are also marked (C and F). In cells treated with lead, disturbances in microfilament pattern were recorded: absence of long bundles twisting around plastids (asterisk), fragmentation of bundles twisting around chloroplasts (red arrow), and thick, local accumulations of actin (red arrowhead). Scale bar = 5 μm
Fig 8.
The analysis of morphology of actin filaments Lemna trisulca mesophyll cells.
Determination of the differences in microfilament morphology was carried out based on the measurement of length (A—fiber length), thickness (B—fiber width) and shape (C—form factor) of microfilament bundles and their branches surrounding chloroplast in control plants and plants treated with lead in darkness. In cells treated with lead, disturbances in microfilaments morphology consisted of shortening (A) and rounding (C) both bundles and their branches. The width of microfilaments was lower in plants treated with lead (B) but only in the case of microfilaments branches. **p < 0.05
Fig 9.
Distribution of hydrogen peroxide in Lemna trisulca.
H2O2 localization by labeling with dichlorodihydrofluorescein diacetate (DCFH-DA; green fluorescence) in fronds (F0—mother fronds and F1—daughter fronds) and in the root of control plants (A) and plants treated with lead (B) in darkness. The control plants showed weak fluorescence in the area of the node (asterisk), sheaths (curly bracket), in the root tip (arrow) and in vascular bundles (arrowhead). Fluorescence intensity was higher in plants treated with lead (B) than in control plants (A). Additionally, under the influence of lead, hydrogen peroxide was generated in mesophyll cells of fronds (double arrow). (C) H2O2 content was also determined spectrometrically in control (Control) plants and plants treated with Pb. **p < 0.05; Scale bar = 1mm
Fig 10.
Colocalization of chloroplasts and Pb in Lemna trisulca.
Lack of relationship between the location of chloroplasts and lead deposits in cell walls of plants treated with the metal in darkness. Lead content is shown as atomic percentage (at%, histograms) and as fragments of X-ray spectra (blue color marks the energy range corresponding to lead atoms) based on X-ray map microanalyses performed in areas of the same size (n = 30). Sample areas of such analyses in regions with chloroplasts (A) and without them (B) are shown as pink rectangles on images from transmission electron microscopy. Below, a diagram of a mesophyll cell section is presented, with marked sample areas of the analyses. Chl—chloroplast, CW—cell wall.