Subcellular Sequestration and Impact of Heavy Metals on the Ultrastructure and Physiology of the Multicellular Freshwater Alga Desmidium swartzii

Abstract

Due to modern life with increasing traffic, industrial production and agricultural practices, high amounts of heavy metals enter ecosystems and pollute soil and water. As a result, metals can be accumulated in plants and particularly in algae inhabiting peat bogs of low pH and high air humidity. In the present study, we investigated the impact and intracellular targets of aluminum, copper, cadmium, chromium VI and zinc on the filamentous green alga Desmidium swartzii, which is an important biomass producer in acid peat bogs. By means of transmission electron microscopy (TEM) and electron energy loss spectroscopy (EELS) it is shown that all metals examined are taken up into Desmidium readily, where they are sequestered in cell walls and/or intracellular compartments. They cause effects on cell ultrastructure to different degrees and additionally disturb photosynthetic activity and biomass production. Our study shows a clear correlation between toxicity of a metal and the ability of the algae to compartmentalize it intracellularly. Cadmium and chromium, which are not compartmentalized, exert the most toxic effects. In addition, this study shows that the filamentous alga Desmidium reacts more sensitively to aluminum and zinc when compared to its unicellular relative Micrasterias, indicating a severe threat to the ecosystem.

Figure 1

Details of cell development of Desmidium in light and electron microscopy. Light microscopic images show the same stages of development as electron micrographs aside. (A) Part of a Desmidium filament with an adult (ac) and a dividing cell (dc). The cell shape is established by a newly-formed primary wall in the isthmus (asterisks); (B) Two adult cells (cross section) connected by primary wall remnants (arrowheads). Isthmus marked by asterisk; (C) Newly-formed primary wall emanating from the isthmus (asterisks) and young septum; (D) Polar view of wall cylinder (arrows) in two developing cells and connecting cell wall (arrowheads); (E) Ultrastructure of dividing Desmidium cells (cross section). The newly-formed primary wall arising from the isthmus (asterisks), and outer and inner cell wall cylinders (arrows) perpendicular to the septum are visible. Remaining cell wall connection between two cells indicated by arrowheads; (F) Splitting of the connecting cell wall visible at the cell edges. On the left side the wall has split only slightly, on the right side more distinctly (arrows); (G) also, the left side has split completely. The arrows now point at the remaining connecting wall; (H) The cell wall within the inner wall cylinder has split as well, recognizable by the appearing background (arrows). N: Nucleus; P: Pores; PW: Primary wall; S: Starch grains; SE: Septum; SW: Secondary wall. Bar = 5 µm.

Figure 1

Details of cell development of Desmidium in light and electron microscopy. Light microscopic images show the same stages of development as electron micrographs aside. (A) Part of a Desmidium filament with an adult (ac) and a dividing cell (dc). The cell shape is established by a newly-formed primary wall in the isthmus (asterisks); (B) Two adult cells (cross section) connected by primary wall remnants (arrowheads). Isthmus marked by asterisk; (C) Newly-formed primary wall emanating from the isthmus (asterisks) and young septum; (D) Polar view of wall cylinder (arrows) in two developing cells and connecting cell wall (arrowheads); (E) Ultrastructure of dividing Desmidium cells (cross section). The newly-formed primary wall arising from the isthmus (asterisks), and outer and inner cell wall cylinders (arrows) perpendicular to the septum are visible. Remaining cell wall connection between two cells indicated by arrowheads; (F) Splitting of the connecting cell wall visible at the cell edges. On the left side the wall has split only slightly, on the right side more distinctly (arrows); (G) also, the left side has split completely. The arrows now point at the remaining connecting wall; (H) The cell wall within the inner wall cylinder has split as well, recognizable by the appearing background (arrows). N: Nucleus; P: Pores; PW: Primary wall; S: Starch grains; SE: Septum; SW: Secondary wall. Bar = 5 µm.

Figure 2

Schematic reconstruction of cytomorphogenesis in Desmidium (cross section). (A) Two adult cells connected by primary wall remnants (arrowheads); (B–D) Morphogenesis by new wall formation; (B) Developing primary wall (red) in the isthmus and outgrowing septum (green); (C) The cell halves are fully separated by a septum; (D) Outer (oc) and inner (ic) cell wall cylinder are developed perpendicular to the septum; (E–H) Morphogenesis by modeling of the newly-formed wall; (E) Connecting cell wall has split (arrowhead); (F) The outer cylinder has fully split and unfolded; (G) The final cell shape is formed at the cell edges; and (H) The remaining connecting wall is split and the inner cylinder is unfolded, only the wall (arrowheads) lying between inner and outer cylinder remains, cytomorphogenesis is completed.

Figure 3

(A–D) Immunogold labeling with different JIM antibodies during cell wall formation in Desmidium; and (E–H) immunogold labeling with BG1 (cross sections). (A) At an early stage the thin septum is labeled by JIM 5; (B) In a subsequent stage septum and newly-formed cell wall cylinders (asterisks) are labeled by JIM 7; (C) A particular population of vesicles in the area of the septum is slightly stained by JIM 8 (arrow). No staining of the septum by JIM 8; (D) Particular vesicle population stained by JIM 13. No staining of the septum by JIM 13; (E) Septum and cell wall cylinders (asterisks) in an early developmental stage. No labeling of the primary wall by BG1; (F) Septum and cell wall cylinders (asterisks) in a later developmental stage. The secondary wall is clearly labeled by BG1, the central part, consisting of the primary wall, remains unstained; (G) Newly-formed primary wall in the isthmus with adjacent secondary wall. The secondary wall is strongly labeled by BG1, the primary wall remains unstained; (H) Secondary wall of a ZnSO₄-treated cell with abnormal depositions (arrow). The secondary wall is labeled by BG1, the abnormal deposition not. PW: Primary wall; SE: Septum; SW: Secondary wall. Bar = 0.5 µm.

Figure 4

Increase in biomass production during exposure of Desmidium filaments to different metals in liquid culture medium for 21 days. Data are the means of three independent experiments ± standard error (SE).

Figure 5

Photosynthetic electron transport efficiency in PSII in controls and heavy metal treated Desmidium filaments after 21 days measured by fast chlorophyll fluorescence.

Figure 6

TEM micrographs of Desmidium. (A) Untreated control; (B) Treated with 10 µM Al₂(SO₄)₃ for 21 days; (C–E) 0.6 µM CdSO₄ for 21 days; (F–G) 10 µM CrVI for 35 days; (H,K) 0.3 µM CuSO₄ for 21 days; and (I,J) 10 µM ZnSO₄ for 21 days; (B) Chloroplast with abnormal dark inclusions (arrows); (C) Altered mitochondria (arrows) and (D,E) autophagosomal-like structures (arrows); (F) Cr-treated cell without visibly affected mitochondria; (G–I) Abnormal depositions on the cytoplasmic site of the cell wall (arrows) induced by Cr (G), Cu (H) and Zn (I); (J) Enlarged mitochondrion after Zn treatment, close to the chloroplast. Cristae are distinctly enlarged and bloated; (K) Vacuole-like structures in the chloroplast (arrows). Cl: Chloroplast; CW: Cell wall; D: Dictyosome; M: Mitochondrion; N: Nucleus; Py: Pyrenoid. Bar = 1 µm, except (D,E) = 0.5 µm.

Figure 7

TEM micrographs of measurement sites (A,E,I) and EEL-spectra measured in Desmidium cells treated with 10 µM Al₂(SO₄)₃ (B–D), with 0.3 µM CuSO₄ (F–H), and with 10 µM ZnSO₄ (J–L), respectively, for 21 days. Measurement areas of the single spectra are indicated as circles in the respective TEM micrographs. The red graphs represent the measurements indicated, the green graphs show control spectra; (B) Al is detected by the K-edge in a precipitation in the secondary cell wall; (C) in dark inclusions in the chloroplast and (D) in starch grains (measurement area not shown in TEM micrograph); (F) Cu is measured by L_2,3-edge in the secondary cell wall; (G) in electron dense inclusion in the chloroplast and (H) in a starch grain; (J) Zn L_2,3-edges were identified in the cell wall itself; (K) in electron dense inclusions of the chloroplast (measurement areas for (J,K) not shown); and (L) In abnormal depositions underneath the secondary cell wall. Bar = 2 µm.

Figure 7

TEM micrographs of measurement sites (A,E,I) and EEL-spectra measured in Desmidium cells treated with 10 µM Al₂(SO₄)₃ (B–D), with 0.3 µM CuSO₄ (F–H), and with 10 µM ZnSO₄ (J–L), respectively, for 21 days. Measurement areas of the single spectra are indicated as circles in the respective TEM micrographs. The red graphs represent the measurements indicated, the green graphs show control spectra; (B) Al is detected by the K-edge in a precipitation in the secondary cell wall; (C) in dark inclusions in the chloroplast and (D) in starch grains (measurement area not shown in TEM micrograph); (F) Cu is measured by L_2,3-edge in the secondary cell wall; (G) in electron dense inclusion in the chloroplast and (H) in a starch grain; (J) Zn L_2,3-edges were identified in the cell wall itself; (K) in electron dense inclusions of the chloroplast (measurement areas for (J,K) not shown); and (L) In abnormal depositions underneath the secondary cell wall. Bar = 2 µm.

Figure 8

Element distribution visualized by electron spectroscopic imaging (ESI) in Desmidium filaments treated with 0.3 µM CuSO₄ (A–C) and 10 µM Al₂(SO₄)₃ (D–F) for 21 days; (A,D) TEM micrographs of Desmidium cells; (B,E) element distribution by ESI in red and (C,F) superimposition of TEM images and corresponding element distribution. Bar = 1 µm.

Figure 9

Detection of the distribution of Al and Zn in Desmidium filaments by morin fluorescence in CLSM, in untreated controls and after exposure of the algae to 50 µM Al₂(SO₄)₃ as well as 10 and 50 µM ZnSO₄, respectively. The first line shows digital interference contrast (DIC) images, the second line presents the morin labeling only, and the third line indicates the merged images (including chloroplast autofluorescence in red). (A–C) Controls show no morin staining; (D–F) Desmidium filaments treated with Al for 1 h show fluorescence of the cell wall; (G–I) After Zn exposure (50 µM) for 1.5 h, pyrenoid regions with surrounding starch grains are labeled (arrows); (J–L) After 21 days treatment with Zn (10 µM), the cell wall and a few spots near the chloroplast are stained (arrows). Bars = 5 µM.