Rescue of heavy metal effects on cell physiology of the algal model system Micrasterias by divalent ions

Abstract

Recent studies have shown that metals such as copper, zinc, aluminum, cadmium, chromium, iron and lead cause severe dose-dependent disturbances in growth, morphogenesis, photosynthetic and respiratory activity as well as on ultrastructure and function of organelles in the algal model system Micrasterias denticulata (Volland et al., 2011, 2012; Andosch et al., 2012). In the present investigation we focus on amelioration of these adverse effects of cadmium, chromium and lead by supplying the cells with different antioxidants and essential micronutrients to obtain insight into metal uptake mechanisms and subcellular metal targets. This seems particularly interesting as Micrasterias is adapted to extremely low-concentrated, oligotrophic conditions in its natural bog environment. The divalent ions of iron, zinc and calcium were able to diminish the effects of the metals cadmium, chromium and lead on Micrasterias. Iron showed most ameliorating effects on cadmium and chromium in short- and long-term treatments and improved cell morphogenesis, ultrastructure, cell division rates and photosynthesis. Analytical transmission electron microscopic (TEM) methods (electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI)) revealed that chromium uptake was decreased when Micrasterias cells were pre-treated with iron, which resulted in no longer detectable intracellular chromium accumulations. Zinc rescued the detrimental effects of chromium on net-photosynthesis, respiration rates and electron transport in PS II. Calcium and gadolinium were able to almost completely compensate the inhibiting effects of lead and cadmium on cell morphogenesis after mitosis, respectively. These results indicate that cadmium is taken up by calcium and iron transporters, whereas chromium appears to enter the algae cells via iron and zinc carriers. It was shown that lead is not taken up into Micrasterias at all but exerts its adverse effects on cell growth by substituting cell wall bound calcium. The antioxidants salicylic acid, ascorbic acid and glutathione were not able to ameliorate any of the investigated metal effects on the green alga Micrasterias when added to the culture medium.

Keywords: AA; Amelioration of metal effects; Antioxidants; Ca; Cd; Cr; EELS; ESI; Fe; Fe-EDTA; Fe-ethylenediaminetetraacetic acid; GSH; Gd; Green algae; HPLC; Heavy metals; Ions; PS II; Pb; ROS; SA; TEM; UPLC-MS; Zn; ascorbic acid; cadmium; calcium; chromium; electron energy loss spectroscopy; electron spectroscopic imaging; gadolinium; glutathione; high-performance liquid chromatography; iron; lead; photosystem II; reactive oxygen species; salicylic acid; transmission electron microscopy; ultra performance liquid chromatography–mass spectrometry; zinc.

Fig. 1

Micrasterias control cell in interphase (a) and fully developed approximately 5 h after mitosis (b); Micrasterias cells after various metals treatments (c–r): 40 μM Pb 4 h (c), 40 μM Pb + 1 mM Ca 4 h (d), 40 μM Pb + 3 μM Gd 4 h (e), 3 μM Gd (30 min) prior to 15 μM Cd 4 h (f), 10 μM Cr, 21 days (g), 1 mM Cr 4 h (h), 10 μM Cr + 100 μM Fe-EDTA, 21 days (i), 1 mM Cr + 100 μM Fe-EDTA 4 h (j), 10 μM Cr + 300 nM Zn, 21 days (k), 1 mM Cr + 300 nM Zn 4 h (l), 600 nM Cd, 21 days (m), 15 μM Cd 4 h (n), 600 nM Cd + 100 μM Fe-EDTA, 21 days (o), 15 μM Cd + 100 μM Fe-EDTA 4 h (p), 600 nM Cd + 300 nM Zn, 21 days (q), 15 μM Cd + 300 nM Zn 4 h (r); Scale bar 100 μm.

Fig. 2

TEM micrographs of Micrasterias from control cells (a and b) and lead treated cells (c and d). Control 4 h (a) and 21 d (b) after mitosis. Short-term treatment (4 h) with 40 μM Pb (c) and long-term treatment (21 d) with 5 μM Pb. No ultrastructural differences between controls and treated cells. C chloroplast, CW cell wall, D dictyosome, M mitochondrium, V vacuole.

Fig. 4

Oxygen turnover (a) and fast electron transport kinetics in PS II (b) after incubation with 5 μM Pb for 21 days.

Fig. 5

Oxygen turnover (a) and fast electron transport kinetics in PS II (b) after 21 days treatment time with 600 nM Cd and 10 μM Cr, with and without 100 μM Fe-EDTA and 300 nM Zn pre-treatment. Cell division rates over the course of 21 days during Cd and Cr treatment with and without Fe-EDTA pre-treatment (c).

Fig. 3

Detail of Micrasterias control cell (a) and ultrastructure, EELS and ESI of 10 μM Cr treated cell after 21 days pre-treatment with 100 μM Fe-EDTA (b–f). Arrow points at Cr induced depositions. Areas of EELS measurements indicated: green area without deposition, red area with deposition (b). EELS measurements of Cr-L_2,3 edge (c), Fe-L_2,3 edge (d). ESI overlay image indicating Cr distribution (e) and Fe distribution (f) in red. C chloroplast, CW cell wall, M mitochondrium, V vacuole.