Effects of lead on the morphology and structure of the nucleolus in the root tip meristematic cells of Allium cepa L

Abstract

To study the toxic mechanisms of lead (Pb) in plants, the effects of Pb on the morphology and structure of the nucleolus in root tip meristematic cells of Allium cepa var. agrogarum L. were investigated. Fluorescence labeling, silver-stained indirect immunofluorescent microscopy and western blotting were used. Fluorescence labeling showed that Pb ions were localized in the meristematic cells and the uptake and accumulation of Pb increased with treatment time. At low concentrations of Pb (1-10 μM) there were persistent nucleoli in some cells during mitosis, and at high concentration (100 μM) many of the nucleolar organizing regions were localized on sticky chromosomes in metaphase and anaphase cells. Pb induced the release of particles containing argyrophilic proteins to be released from the nucleus into the cytoplasm. These proteins contained nucleophosmin and nucleolin. Pb also caused the extrusion of fibrillarin from the nucleus into the cytoplasm. Western blotting demonstrated the increased expression of these three major nucleolar proteins under Pb stress.

Figure 1

Confocal laser scanning microscope micrographs from root tip meristematic cells of A. cepa exposed to Pb(NO₃)₂ for 4, 24 and 48 h, using Leadmium™ Green AM dye (Invitrogen). (A1,B1,C1,D1) Pb detection; (A2,B2,C2,D2) Bright field image; (A3,B3,C3,D3) Merged image of “Pb detection” and “bright field image”. All images are taken at ×10 magnification, and green fluorescence represents the binding of the dye to Pb. One of six typical examples is selected for each treatment. Scale bars = 100 μm. M = meristematic zone. RC = root cap.

Figure 2

Effects of different concentrations of Pb on nucleolar organizing regions (NORs) in root tip meristematic cells of A. cepa during mitosis. (a–h) Normal mitotic process. (a) The interphase cell; (b,c) Showing decondensed chromatin fibers were around the nucleoli; (d) Showing that the nucleoli disappeared in their characteristic structures; (e) Showing NORs localized on metaphase chromosomes; (f) Showing NORs migrated with the chromosomes to the poles at anaphase; (g) Showing nucleoli rebuilt at early telophase and the size increased; (h) Showing the two daughter nuclei entered interphase, and mitosis was completed; (i–l) Mitotic process under Pb stress. (i,j) Showing persistent nucleoli during metaphase ((i) 10 μM Pb, 24 h; (j) 1 μM Pb, 48 h);(k) Showing sticky chromosomes with Ag-stained NOR particles at metaphase (100 μM Pb, 48 h);and (l) Showing sticky chromosomes with Ag-stained NOR particles at anaphase (100 μM Pb, 48 h).Scale bars = 10 μm. Nucleoli and NORs: dark brown; nuclei and chromosomes: green; cytoplasm: yellow. Arrowhead shows NORs.

Figure 3

Effects of different concentrations of Pb on nucleoli in the root tip meristematic cells of A. cepa. (a) Control cell; (b,c) Silver-stained particles containing argyrophilic proteins in nucleus (10 μM Pb, 48 h); (d) Silver-stained particles accumulated in nucleus (100 μM Pb, 24 h); (e,f) Nucleolus particles extruded from the nucleus into the cytoplasm ((e) 10 μM Pb, 72 h; (f) 100 μM Pb, 48 h); (g,h) Nucleolar material in the cytoplasm ((g) 10 μM Pb, 72 h; (h) 100 μM Pb, 72 h); (i) Irregular nucleus (100 μM Pb, 48 h). Scale bars = 10 μm. Nucleoli and nucleolar material: dark brown; Nuclei: green; cytoplasm: yellow. Arrowheads show silver-stained particles.

Figure 4

Simultaneous detection of nucleophosmin after incubation with primary anti-nucleophosmin antibody and secondary antibody conjugated with FITC (Fluorescein isothiocyanate) (green), and of DNA after incubation with DAPI (4',6-diamidino-2-phenylindole) (blue) in the same single optical section using confocal microscopy. (A1,B1,C1,D1) Nucleophosmin detection; (A2,B2,C2,D2) DNA detection; (A3,B3,C3,D3) Merged image of “nucleophosmin detection” and “DNA detection”; (A4,B4,C4,D4) bright field image. (A1–A4) Nucleophosmin mainly localized in the nucleolus of control cells; (B1–B4) The migration of nucleophosmin from the nucleolus to the nucleoplasm in cells treated with 100 μM Pb for 24 h; (C1–C4) The movement of nucleophosmin from the nucleoplasm into the cytoplasm in cells treated with 100 μM Pb for 48 h; and (D1–D4) Nucleophosmin in the cytoplasm of cells treated with 100 μM Pb for 72 h. Scale bars = 10 μm. Arrowhead shows nucleolus.

Figure 5

Simultaneous detection of nucleolin after incubation with primary anti-nucleolin antibody and secondary antibody conjugated with TRITC (Tetramethylrhodamine isothiocyanate) (red), and of DNA after incubation with DAPI (blue) in the same single optical section using confocal microscopy. (A1,B1,C1,D1) Nucleolin detection; (A2,B2,C2,D2) DNA detection; (A3,B3,C3,D3) Merged image of “nucleolin detection” and “DNA detection”; (A4,B4,C4,D4) bright field image. (A1–A4) Nucleolin in nucleolus of control cells; (B1–B4) The migration of nucleolin from the nucleolus to the nucleoplasm in cells treated with 100 μM Pb for 24 h; (C1–C4,D1–D4) Nucleolin in the cytoplasm of cells treated with 100 μM Pb for 48 h (C1–C4) and 72 h (D1–D4). Scale bars = 10 μm. Arrowhead shows nucleolus.

Figure 6

Simultaneous detection of fibrillarin after incubation with primary anti-fibrillarin antibody and secondary antibody conjugated with FITC (green), and of DNA after incubation with DAPI (blue) in the same single optical section using confocal microscopy. (A1,B1,C1,D1) Fibrillarin detection; (A2,B2,C2,D2) DNA detection; (A3,B3,C3,D3) Merged image of “fibrillarin detection” and “DNA detection”; (A4,B4,C4,D4) bright field image. (A1–A4) Fibrillarin in the nucleolus of control cells; (B1–B4) Migration of fibrillarin from the nucleolus to the nucleoplasm or the cytoplasm in the cells treated with 100 μM Pb for 24 h; (C1–C4) Fibrillarin in the cytoplasm of the treated cells at 48 h, and 72 h (D1–D4). Scale bars = 10 μm. Arrowhead shows nucleolus.

Figure 7

Effect of Pb (100 μM Pb, 48 h), on the expression of nucleophosmin, nucleolin and fibrillarin. Bands were detected by western blotting (A) and their intensities were measured by “Quantity One” software (Bio-Rad, Berkeley, CA, USA) (B). Bar indicates standard error (SE). n = 5. p < 0.05.