Copper is an essential metal for normal plant growth and development, although it is also potentially toxic. Copper participates in numerous physiological processes and is an essential cofactor for many metalloproteins, however, problems arise when excess copper is present in cells. Excess copper inhibits plant growth and impairs important cellular processes (i.e., photosynthetic electron transport). Since copper is both an essential cofactor and a toxic element, involving a complex network of metal trafficking pathways, different strategies have evolved in plants to appropriately regulate its homeostasis as a function of the environmental copper level. Such strategies must prevent accumulation of the metal in the freely reactive form (metal detoxification pathways) and ensure proper delivery of this element to target metalloproteins. The mechanisms involved in the acquisition of this essential micronutrient have not been clearly defined although a number of genes have recently been identified which encode potential copper transporters. This review gives a briefly overview of the current understanding of the more important features concerning copper toxicity and tolerance in plants, and brings information of recent findings on copper trafficking including copper detoxification factors, copper transporters and copper chaperones.

Copper in plants

Germinating seedlings of mung bean (Phaseolus vulgaris L. cv. K-16) were treated with different concentrations of cadmium acetate (10, 50 and 100 μM). Cd2+ lowered the chlorophyll and heme levels. The level of lipid peroxides were higher on day 3 than on day 6. However, Cd2+ treatment significantly enhanced the level of lipid peroxides. Similarly, a dose-dependent induction of lipoxygenase (EC 1.13.11.12) activity was observed with Cd2+ treatment. Further, the activities of antioxidant enzymes such as superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6) were decreased. Our results suggest that lipoxygenase-mediated accumulation of lipid peroxides on the one hand and inhibition of free radical scavenging enzymes like superoxide dismutase and catalase on the other caused a pronounced reduction in the chlorophyll and heme levels of the seedlings. The experiments conducted on the effect of Cd2+ on dark-grown seedlings did not conform with the result of light-grown seedlings. Though chlorophyll and heme levels decreased in a dose-dependent manner, no accumulation of lipid peroxides was observed, suggesting that the inhibition of chlorophyll synthesis by Cd2+ is achieved both by reaction with constituent biosynthetic enzymes as well as peroxide-mediated degradation.

Phytotoxicity of cadmium ions on germinating seedlings of mung bean (Phaseolus vulgaris): Involvement of lipid peroxides in chlorphyll degradation

Following experiments which studied the substitution of the central ion of isolated chlorophylls by heavy metal ions in vitro, in vivo experiments with submersed water plants were carried out. It was discovered that the substitution of the central atom of chlorophyll, magnesium, by heavy metals (mercury, copper, cadmium, nickel, zinc, lead) in vivo is an important damage mechanism in stressed plants. This substitution prevents photosynthetic light-harvesting in the affected chlorophyll molecules, resulting in a breakdown of photosynthesis. The reaction varies with light intensity. In low light irradiance all the central atoms of the chlorophylls are accessible to heavy metals, with heavy metal chlorophylls being formed, some of which are much more stable towards irradiance than Mg-chlorophyll. Consequently, plants remain green even when they are dead. In high light, however, almost all chlorophyll decays, showing that under such conditions most of the chlorophylls are inaccessible to heavy metal ions.

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Environmental relevance of heavy metal-substituted chlorophylls using the example of water plants

Summary
1. All heavy metals, including those that are essential micronutrients (e.g. copper, zinc, etc.), are toxic to algae at high concentrations.

2. One characteristic feature of heavy-metal toxicity is the poisoning and inactivation of enzyme systems. Many of the physiological and biochemical processes, viz., photosynthesis, respiration, protein synthesis and chlorophyll synthesis, etc., are severely affected at high metal concentrations.

3. Some algae inhabit waters chronically polluted with heavy-metal-laden wastes from mining and smelting operations; Nodularia sp., Oscillatoria sp., Cladophora sp., Hormidium sp., Fucus sp. and Laminaria sp., etc., occur in metal-rich waters. These algal forms are probably more capable of combating the toxic levels of heavy metals and this attribute is a result of physiological and/or genetic adaptations. The sensitivity or tolerance to heavy metals varies amongst different algae. The phenomena of multiple tolerance and co-tolerance may be exhibited by some algae.

4. Heavy-metal pollution causes reduction in species diversity leading to the dominance of a few tolerant algal forms. The primary productivity also decreases after metal supplementation.

5. The uptake and accumulation of heavy metals can be active (energy-dependent), passive (energy-independent), or both.

6. Heavy metals can be safely stored as intranuclear complexes by some algae. Notwithstanding this, some changes in the cell wall can enable the algae to tolerate heavy metals by checking the entry of the metals (exclusion mechanism).

7. The metal content of algae growing in a waterbody may yield valuable information for simulating heavy metal pollution: several species of Cladophora and Fucus have been extensively used for this purpose.

8. Several factors affect and determine toxicity of heavy metals to algae. At low pH, the availability of heavy metals to algae is greatly increased, as a consequence of which pronounced toxicity is evident. Hard waters decrease metal toxicity. Some ions, e.g., calcium, magnesium and phosphorus, can alleviate toxicity of metals.

9. The presence of other metals can influence toxicity of a heavy metal through simple additive effect or by synergistic and antagonistic interactions. Similarly, other pollutants can influence heavy-metal toxicity.

10. The toxicity of heavy metals depends upon their chemical speciation. Various ionic forms of a metal characterized by different valency states, may be differentially toxic to a test alga.

11. Amino acids, organic matter, humic acids, fulvic acid, EDTA, NTA, etc. can complex with heavy metals and render them unavailable. This may eventually lead to less toxicity.

12. Heavy-metal toxicity largely depends upon algal population density: the denser the population the more numerous the cellular sites available, leading to decreased toxicity.

Phycology and heavy-metal pollution

Inhibition of photosynthesis by heavy metals is well documented. In this review the results are compared between in vitro experiments on isolated systems (chloroplasts, enzymes -.), experiments on excised leaves and intact plants and algae in vivo. In vitro experiments suggest potential sites of heavy metal interaction with photosynthesis at several levels of organisation, which are not necessarily confirmed in vivo. Analytical data on subcellular heavy metal level are generally missing to discuss their mechanism of action in the intact organism. In the field factors such as soil characteristics and air pollution have to be taken into account for assessing the mechanism of action of heavy metals on photosynthesis in plants, growing in a polluted erea.

Inhibition of photosynthesis by heavy metals.

Effects of copper on photosynthetic electron transport systems in spinach chloroplasts

Copper strongly inhibited 2,6-dichloroindophenol (DCIP) photoreduction in the broken cells of the green alga Ankistrodesmus falcatus (C303), and the activity lost could not be restored by adding 1,5-diphenylearbazide (DPC). Inactivation of the DCIP Hill reaction reached 45% after incubation with 10 μM cupric sulfate for 20 min. In the same time, copper (13 μg/mg chlorophyll) was bound to the broken cells. Addition of 10 mM KCl reduced copper binding by about 53%. Fluorescence intensity at room temperature decreased upon addition of cupric sulfate and was partially restored by adding 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), These results suggest that copper inactivates electron transport between the oxidizing side of the reaction center of photosystem II and the electron-donating site of DPC. Further, the effect of light intensity shows that copper mostly affected the reaction rate of the dark step and had less inhibitory effect on the quantum efficiency of the primary reaction of electron transport in photosystem II.

Hiroko Tamai

Papers

Effects of copper on photosynthetic electron transport systems in spinach chloroplasts

Inhibition of Photosystem II in the Green Alga Ankistrodesmus falcatus by Copper

Purification and characterization of δ-aminolevulinic acid dehydratase from Chlorella regularis

Studies on chlorophyllase of Chlorella protothecoides IV. Some properties of the purified enzyme

A simple purification method for the preparation of solubilized chlorophyllase from Chlorella protothecoides