Lead and copper effects on lipid metabolism in cultured lichen photobionts with different phosphorus status

Abstract

We evaluated the ability of heavy metals (copper, lead) to alter lipid metabolism in four algal lichen photobionts following short term exposure. Metal concentrations (10 μM) were equivalent to environmentally relevant levels that have been reported to have effects on intact algae. The algae were grown under normal or deficient phosphate conditions to assess any interactions with the heavy metal stress. Given the frequent sensitivity of lichens to copper and lead, there were surprisingly small changes on lipid metabolism, as assessed by radiolabelling from [1-¹⁴C]acetate. The main effects, which were seen in a number of cases, were an overall inhibition of total lipid labelling and a relative increase in the labelling of triacylglycerols in the non-polar fraction. Both of these changes can be viewed as reflecting general toxicity of heavy metals. The Coccomyxa photobiont species were more sensitive than Trebouxia species, which fits with the general distribution of the latter in lichens inhabiting harsh environments.

Introduction

Lichens are symbiotic associations between heterotrophic fungi (mycobiont) and photosynthetic prokaryotic (cyanobacterium) or eukaryotic (alga) organisms referred to as a photobiont. Lichens are important components of the vegetation of many ecosystems in the world from the tropics to the polar regions (approximately 8% of terrestrial ecosystems are lichen-dominated). They include about 17,000 species and are often especially important in extreme environments (Nash, 1996a, Hale, 1983). Around 85% of lichen-forming fungi are symbiotic with green algae, approximately 10% with cyanobacteria (blue-green algae) and 3–4% are cephalodiate species which associate simultaneously with cyanobacteria and green algae (Honegger, 1996).

Lichens are useful as biomonitors of environmental pollution, including raised heavy metal levels for several reasons. First, they have a wide geographic distribution and as perennial, slow-growing and long-lived organisms, maintain a fairly uniform morphology over a long period of time (Ahmadjian, 1993). Second, lichens can accumulate many compounds in high concentrations due to the absence of a wax cuticle and stomata on the surface of lichen thalli that allows diffusion of contaminants into the tissue. Third, because lichens lose water as a result of evaporation during dry periods this may lead to pollutant concentration and thus increase lichen sensitivity. Fourth, photosynthesis (which is often a primary target of pollution) can occur at low temperatures in lichens which are, thus, sensitive throughout the year (Nash, 1996b, Gries, 1996). Finally, the sensitivity of lichens to pollution may also be related to their symbiotic nature that requires that a metabolic balance between symbionts is maintained and a disturbance may readily lead to a breakdown of the whole association (Gries, 1996).

Several reviews (e.g., Tyler, 1989, Puckett and Burton, 1981, Garty, 1993) have dealt with aspects of accumulation, tolerance, and toxicity of heavy metals in lichens. Generally, reductions in photosynthetic and respiration rates are found, often with increased membrane permeability and degradation. With some lichens additional, more specific, metabolic responses have been noted (Puckett, 1976, Brown and Beckett, 1983, Brown, 1995, Branquinho et al., 1997b). The photobiont and fungal components of the lichens may respond differently (Tarhanen, 1998). However, the photobionts are clearly key components because of their role in photosynthesis (Ahmadjian, 1993) and, also, because molecular mechanisms causing sensitivity are often confined to the photobiont (e.g., Pawlik-Skowrońska et al., 2002).

The specific effects of heavy metals on photosynthesis has been studied in algae and cyanobacteria where Cu, Ni, Pb are all compounds which can cause inhibition at environmentally-relevant concentrations (Nalewajko and Olaveson, 1995, Rai et al., 1995, Rai et al., 1996, Rai and Rai, 1997, Lu and Zhang, 1999). Other membrane-located activities which are also adversely affected by heavy metal exposure are cation leakage (Lage et al., 1996), ATP production (Rai and Rai, 1997) and ion uptake (Nalewajko and Olaveson, 1995).

Lipids are key components of membranes and, therefore, are vital for their function (Murata and Siegenthaler, 1998). Furthermore, lipid metabolism is known to be affected by environmental stress and, in resistant organisms, to be able to adjust appropriately (see Thompson, 1996, Harwood, 1998, Rama Deli and Prasad, 1999). Following heavy metal stress, several aspects of lipid biochemistry have often been noted to change. These include qualitative and quantitative alterations in lipids, inhibition of biosynthetic pathways and a reduction in unsaturated fatty acids due to metal-enhanced peroxidation (Harwood, 1994, Rama Deli and Prasad, 1999, Dietz et al., 1999).

Although abundant information on lipids of metal-stressed higher plants is available (for review see Rama Deli and Prasad, 1999), much less is known of the effects of heavy metals on algal or cyanobacterial lipids. Matson et al. (1972) showed the drastic reduction in galactolipid biosynthesis, via an inhibition of galactosyl-transferase activity, in two green algae (Monoraphidium braunii, Euglena gracilis) treated with mercury. Effects of cadmium on Euglena gracilis membrane lipids resulted in a lowering of sterol content but an increase in phosphatidylglycerol and cardiolipin in light-exposed cells (Einicker-Lamas et al., 1996). Similarly, cells of the marine diatom Asterionella glacialis treated with mercury and cadmium also had decreased sterols and their total polyunsaturated fatty acid content was reduced (Jones et al., 1987). Exposure to heavy metals (Cu, Zn and Cd) also changed fatty acid patterns in Selenastrum capricornutum (McLarnon-Riches et al., 1998) and fatty acid synthesis, as estimated by labelling from [¹⁴C]acetate, in the marine brown algae, Fucus serratus and F. vesiculosus (Harwood and Jones, 1989).

It should be noted, however, that eukaryotic algae use phytochelatins to complex heavy metals whereas cyanobacteria may utilize metallothioneins. So the exact molecular mechanisms to ensure some resistance to heavy metals exposure may be somewhat different. Nevertheless, the response of algae, cyanobacteria and lichens to heavy metals, such as copper, is often very similar (Bačkor et al., 2004).

Some complex interactions between other nutrients and heavy metal toxicity have been well documented, including phosphorus status. Thus, the efficiency of phosphate utilization or the ability to tolerate phosphate deficiency often influences metal sensitivity in many plants and algae (Foy et al., 1978, Rai et al., 1981). Cd has been reported to inhibit phosphate uptake in Anacystis nidulans (Singh and Yadav, 1984), while Al affected only its mobilization in Anabaena cylindrical (Pettersson et al., 1988). Conversely, the important role of phosphate in regulating cellular uptake of copper has been established using the nutritionally starved cyanobacterium Nostoc calcicola (Verma et al., 1991) and for Zn and Cd in Chlorella autotrophica (Wang and Dei, 2001). In a following study, Verma et al. (1993), presented evidence that Cu toxicity in cyanobacteria was due to the Cu-induced phosphate starvation and that exogenous addition of phosphate could antagonize the Cu-effect in N. calcicola (Verma et al., 1993). Phosphorus metabolism was also shown to influence cadmium toxicity to N. linckia (Husaini and Rai, 1991) and a significant amelioration of Cr and Pb toxicity has been observed in Nostoc muscorum at high concentrations of phosphate (Singh et al., 1993). Some of these interactions may include involvement of polyphosphate granules or bodies (PPB) in intracellular metal accumulation. These granules may maintain low cytoplasmic levels of metals, thus reducing their toxicity, as has been demonstrated in the diatoms Amphora and Navicula exposed to copper (Daniel and Chamberlain, 1981). In the cyanobacterium Nostoc muscorum, PPB have been suggested to be the main sink for nickel (Singh et al., 1992). Moreover, a copper-resistant Anabaena variabilis strain contained more PPB and also had higher internal phosphorus levels than the sensitive strain (Hashemi et al., 1994) while Torres et al. (1998) showed Zn, Pb, Mg and Al sequestering to be important in the PPB of Plectonema boryanum.

Unfortunately, there have been few studies of the effect of metals on lichen photobionts. Backor et al. (1998) studied the effect of metals (Cd, Cu, Hg) on the lichen photobiont, Trebouxia irregularis while copper tolerance was evaluated in Trebouxia erici (Bačkor and Váczi, 2002). The same group also studied the effect of the above metals on chlorophyll levels in Coccomyxa sp. and Myrmecia biatorellae (Bačkor and Dzubaj, 2004).

At the same time, heavy metals, like Pb and Cu, are important industrial pollutants and their environmental effects may be increasing (Branquinho et al., 1997b, Maksymiec, 1997). Because of the importance of lichens in the environment and their reported sensitivity to heavy metals, we wished to know more about the biochemistry of Cu and Pb on lipid metabolism. In order to provide a simpler system for the experiments we used cultured algal photobionts and examined the action of Cu and Pb in organisms of different phosphorus status. The heavy metal levels used were chosen because they were reported to be toxic to lichens but, surprisingly, they caused relatively little immediate effect on the photobionts alone.