Ultrastructural changes in Chlamydomonas acidophila (Chlorophyta) induced by heavy metals and polyphosphate metabolism

Abstract

Ultrastructural changes induced by heavy metals (cadmium, zinc, and copper) and polyphosphate metabolism were studied in Chlamydomonas acidophila. Transmission electron microscopy indicated that cadmium led to the most drastic morphometric changes. An increase in number and volume of starch grains and vacuoles as well as the presence of electron dense deposits in vacuole and membrane whorls were observed. Energy-dispersive X-ray analysis revealed that vacuolar deposits inside cells treated with cadmium contained phosphate and cadmium. These ultrastructural changes were accompanied by a change in the intracellular polyphosphate level, as shown by in vivo ³¹P-nuclear magnetic resonance. It was also observed that cadmium treatment caused polyphosphate degradation and increased vacuolar short-chains and orthophosphates.

1 Introduction

Acidification of the environment by acid rain has adverse effects on ecological systems. Some reports show that a shift in pH from neutral to acidic causes a reduction in algal and fish population. Sulfuric acid directly results in poor production of fish eggs, with production of spines and other deformities [1,2]. Indirectly, lower pH induced by acid rain is accompanied by an increase in metal ion concentration in soil and aquatic environments and acidification leads to leaching of poisonous metals such as aluminum, cadmium and mercury, from the soil. A close relationship exists between metal toxicity and pH in soil and aquatic environments.

Chlamydomonas acidophila, isolated from Lake Katanuma in Japan, has adapted to acidic environmental stress (acidification and heavy-metal toxicity) [3]. In particular, we studied the mechanism of heavy-metal detoxification in C. acidophila, because heavy-metal contamination of the environment is a severe problem with consequences throughout the food chain.

Heavy-metal concentrations in the environment have increased due to industrial activity and acid rain. For example, Cd usage has increased in plastic manufacturing, electroplating of steel, Ni–Cd batteries and in pigments [4].

In this study, the mechanism of heavy-metal detoxification was studied in C. acidophila by observing ultrastructural damage. Additionally, we focused on phosphate metabolism and metal detoxification because energy-dispersive X-ray (EDX) analysis on C. acidophila suggested a close relationship with metal detoxification.

We estimated ultrastructural damage using morphometric analysis. Electron microscope morphometric techniques can be used to quantitatively assess the impact of perturbations on cytological characteristics in their natural environment [5]. The detoxification of heavy metals is accomplished by several different cellular mechanisms: exclusion, precipitation, reduction, and active transport. This alga may have another detoxification mechanism, in addition to the metallothionein and phytochelatin. This mechanism will help the understanding of adaptation to acidified environments in other organisms.

2 Materials and methods

2.1 Culture condition

Stocks of the green alga, C. acidophila (Chlorophyta), were grown as previously described [3]. Axenic cultures were maintained by periodic transfers in modified Sager–Granick Medium [6] (1.22 mM MgSO₄·7H₂O, 3.75 mM NH₄NO₃, 0.57 mM K₂HPO₄, 0.73 mM KH₂PO₄, 0.36 mM CaCl₂, 37 μM FeCl₃·6H₂O, 16.2 μM H₃BO₄, 3.5 μM ZnSO₄·H₂O, 2.02 μM MnCl₂·4H₂O, 0.24 μM CuSO₄·5H₂O, 0.84 μM CoCl2·6H₂O, 0.83 μM Na₂MoO₄·H₂O, 50 mM succinic acid, with pH adjusted to 4.0 by addition of 1 N HCl). Cells were grown at 20°C, with 146 mE m⁻² s⁻¹ of illumination and a 12 h/12 h light/dark cycle. All glassware was washed with HCl and media and glassware were sterilized by standard autoclave procedures.

2.2 Metal treatment

Effective concentrations of cadmium, copper and zinc were achieved by reducing the population growth of C. acidophila to 50% of the control values (EC₅₀) obtained from the 72-h static exposure tests. Static metal treatment was carried out for 3 days with the addition of EC₅₀ concentration, as previously reported [3]. In short, a modified Sager–Granick medium, pH 4, was used for the metal treatment with the addition of the following nominal metals at the concentrations: 20 μM 3CdSO₄·6H₂O, 200 μM CuCl₂·2H₂O, and 1.5 mM ZnCl₂. Cells were grown as described above.

2.3 Morphometric analysis

For studies where techniques of morphometric analysis were used, the cultures were exposed for 72 h to three metals as described above. After the metal treatment, the cells were harvested and fixed with 1.0% glutaraldehyde in 50 mM acetate buffer for 20 min at room temperature. Fixed samples were washed 10 times with acetate buffer (pH 4) and post-fixed with 2% OsO₄ overnight at 4°C.

Samples were washed with acetate buffer, dehydrated in a graded ethanol series and embedded in Epon 812. Ultrathin sections were stained in aqueous 1% (w/v) uranyl acetate and lead citrate. Transmission electron micrographs (TEM) were obtained by JEOL-1230 electron microscope using an accelerating voltage of 60 kV. The sections of a cell were selected at random and photographed.

Following general techniques for modified morphometric analysis [7–11], cellular features, including whole cell, cytoplasm, chloroplast, vacuole, starch grains, pyrenoid, nucleus and mitochondria, have been analyzed. Over 20 microphotographs were scanned (Epson GT-7200U), each representing a separate cell, and analyzed (NIH image program) to determine the area of each organelle for each of the test conditions. The mean and standard error were obtained for each measurement and significance of the differences was determined by one-way analysis of variance (ANOVA).

2.4 Accumulation of Cd

Accumulations of Cd were measured using an Atomic Absorption Spectrophotometer (AA-660, Shimazu). Cells, treated with 10 or 20 μM Cd for 1, 2, and 3 days, were collected by centrifugation and washed three times with 0.1 M ethylenediamine tetraacetic acid (EDTA). Samples were dried and digested with a mixed acidic solution of HNO₃ and HClO₄ (9:1) for 12 h at 100°C. Samples were diluted with 1 N HCl and analyzed for Cd content. Protein levels were analyzed using the BCA method (Shigma).

2.5 EDX analysis

EDX analysis was performed at the electron microscopy center of Tokyo Metropolitan University. Specimen grids were examined by a Hitachi H 710 FA transmission electron microscope at an accelerating voltage of 80 kV. Fine probe size was adjusted to cover the vacuolar deposits, and X-rays were collected for 100 s utilizing a thin window detector. Carbon-coated copper grids supported the thin sections.

2.6 ³¹P-NMR (nuclear magnetic resonance) measurement

³¹P-NMR measurements were performed using a Varian XL-400 spectrometer operated at 162 MHz with a sweep width of 32 362 Hz. The free induction decay was performed with a line broadening of 10 Hz. Pulses with a flip angle of 60° and repetition rate of 0.5 s were used. The cells were measured at room temperature in a 5-mm-diameter probe head. All chemical shifts were measured in ppm using triphenylphosphate as an external standard. Chemical shifts were measured relative to an external standard of 85% orthophosphoric acid. It is reported that the ratio of polyphosphate to orthophosphate in vacuoles is growth-dependent [12]; it was measured in 6-day-old (exponential phase) cells. Cells in the exponential phase of growth were transferred to a medium containing 20 μM Cd for 3 days (Cd-treated) and then retransferred to a non-metal medium for 3 days (recovered cells). Measurements were performed on untreated cells, Cd-treated cells and recovered cells. Cells were harvested by centrifugation, washed in phosphate-free 50 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) at pH 4.0, and suspended in the same buffer to a cell concentration of approximately 10⁹ cells ml⁻¹.

3 Results

3.1 Effects of heavy metals on ultrastructure

The structural damage due to exposure to each metal was distinctly different. When treated with Cd, the whole cell size increased compared to untreated cells (Fig. 1a,b). On the other hand, cells treated with Cu and Zn decreased in size while maintaining their ellipsoidal shape (Fig. 1c,d).

Detailed morphometric analyses of the Cd, Cu and Zn treatments are presented in Table 1. As shown therein, 20 μM Cd caused the most drastic morphometric changes. The sizes of starch granules, vacuoles and chloroplasts were increased 2.43-fold (P<0.01), 1.91-fold (P<0.05) and 1.66-fold (P<0.01), respectively. Conversely, pyrenoid structures were noticeably reduced in size to 46% of that of the controls. In addition, a new structure was formed after Cd treatment, consisting of non-membranous, electron-dense deposits, observed in vacuoles. This vacuolar deposit was of undefined shape and often occupied almost the whole vacuole (Fig. 1b). Vacuolar deposits were also observed in Cu- and Zn-treated cells (Fig. 1c,d).

All organelles decreased in size in Cu-treated cells. Although the size of the whole cell in Zn-treated cells decreased to 85% of that of the control cells, the size of starch grains of Zn-treated cells increased slightly. The presence of membrane whorls (MW) was observed in the Cu- and Zn-treated cells (Fig. 1c,d).

3.2 Accumulation of Cd

The presence of Cd clearly caused drastic ultrastructural changes (Fig. 1b). Cd accumulation within cells increased with time and the concentration was the highest in cells treated with 20 μM Cd for 3 days (Fig. 2). Cells were washed with EDTA before analysis and the obtained values represented intracellular Cd accumulations.

3.3 Localization of Cd

X-ray microanalysis was performed to evaluate the local elemental distribution within the cell. The electron probe was focused on several analytical points including electron-dense deposits in vacuoles. An arrow in Fig. 3a shows one of the analytical points. In the EDX spectrum, signals of Cd and phosphate are clearly observed (Fig. 3b). Other signals represent Cu from the grid and Pb from the stain solution. Cd was also detected in the cell membrane. We confirmed that these electron-dense deposits contained both phosphate and Cd.

3.4 Effect of Cd on polyphosphate metabolism

Fig. 4 shows the effect of Cd stress on polyphosphate metabolism in C. acidophila as revealed by ³¹P-NMR. After Cd treatment for 3 days, changes in the intensity of the polyphosphate peak and the vacuolar phosphate peak were apparent. The polyphosphate peak disappeared almost completely, accompanied by an intense increase in the vacuolar phosphate peak (Fig. 4a), suggesting that the two processes were related. To determine whether the disappearance of polyphosphate is reversible or not, Cd-treated cells were re-incubated in modified Sager–Granick medium for 3 days, and a ³¹P-NMR spectrum was taken. Fig. 4c shows that the polyphosphate peak recovered to the previous level. In addition, a decrease in the vacuolar phosphate peak and the sugar phosphate peak increased. The phosphate level in the recovered cells did not return to the original level.

4 Discussion

The three studied metals influenced C. acidophila cells differently, with the greatest ultrastructural change caused by cadmium. Although EC₅₀ is one index for biological toxicity, it cannot reveal the exact cytological toxicity. Morphometric analysis at the ultrastructural level may provide ecologically valuable insights. This change involved enlargements of starch granules and vacuoles, a reduction in pyrenoid and mitochondrial size (Table 1) and the presence of MW and vacuolar deposits (Fig. 1b–d).

Similar increases in the starch granule size and a reduction in mitochondrial size were reported in Chlorella cells, in which the rapid deterioration of mitochondria caused accumulation of starch grains [13]. Mitochondria are a primary target of the cadmium-associated cytotoxicity in freshwater green algae [14]. Consequently, since the respiratory activities cannot be carried out without mitochondria, starch accumulation results in the disarrangement of the chloroplasts.

Rachlin et al. [7] detected accumulation of metals (Ni, Cu, Co, Zn, Cd, and Hg) in MW in Plectonema boryanum and suggested that this new structure was detached from residues of thylakoids for a cellular detoxification mechanism.

Enlargement of vacuoles and deposits inside the cells were the most remarkable changes but the shape of the deposits was difficult to define clearly. C. acidophila accumulated a large amount of Cd within cells (Fig. 2) and vacuolar deposits included phosphate and cadmium traces (Fig. 3b). Many researchers have reported that electron-dense deposits were from polyphosphate bodies. Polyphosphate bodies have the ability to accumulate metals and also to protect algal cells from metal toxicity [8,15].

The exact role of vacuoles in heavy-metal detoxification is not yet clear, but vacuolation could contribute to compartmentalization of toxic metals, as suggested by Davies et al. [15]. In yeasts and fungi a large proportion of the accumulated ions is located inside the vacuole in an ionic form or bound to the polyphosphates of lower molecular mass [16]. During our investigation, the peak of polyphosphate disappeared almost completely with an intense increase in the vacuolar phosphate peak (Fig. 4b) after Cd stress.

Inorganic polyphosphates are linear polymers of many orthophosphate (P_i) residues linked by high-energy phosphoanhydride bonds and are found in cells of all organisms [17]. This observation indicates that polyphosphate hydrolyzed, forming shorter-chain species or orthophosphates and combined with Cd in vacuoles. Recent studies have pointed out that the degradation of polyphosphate into orthophosphate is more important for heavy-metal tolerance than the polyphosphate level in the cells [18–20]. Cells that had recovered from the Cd stress simultaneously increased the peaks of cytoplasmic and sugar phosphate. A vacuolar phosphate peak remained (Fig. 4c) and recovered cells did not return to their former phosphate level. Under osmotic stress, a reversible precursor–product relationship existed between cytoplasmic phosphate and polyphosphate in mycelium of Neurospora crassa[21]. However, under Cd stress, the vacuolar phosphate, combined with Cd in the recovered cells, could not convert to cytoplasmic phosphate. Vacuolar phosphate–Cd must be the final state for metal detoxification.

In this study we have confirmed that the role of vacuoles and polyphosphate degradation is important in heavy-metal detoxification.

Acknowledgements

The authors are grateful to Dr. Adriana Paulovicova (Ochanomizu University) for general support and the English text revision.

References