Mercury Toxicity in Plants.
Mercury poisoning has become a problem of current interest as a result of environmental pollution on a global scale. Natural emissions of mercury form two-thirds of the input; manmade releases form about one-third. Considerable amounts of mercury may be added to agricultural land with sludge, fertilizers, lime, and manures. The most important sources of contaminating agricultural soil have been the use of organic mercurials as a seed-coat dressing to prevent fungal diseases in seeds. In general, the effect of treatment on germination is favorable when recommended dosages are used. Injury to the seed increases in direct proportion to increasing rates of application. The availability of soil mercury to plants is low, and there is a tendency for mercury to accumulate in roots, indicating that the roots serve as a barrier to mercury uptake. Mercury concentration in aboveground parts of plants appears to depend largely on foliar uptake of [Hg.sup.0] volatilized from the soil. Uptake of mercury has been found to b e plant specific in bryophytes, lichens, wetland plants, woody plants, and crop plants. Factors affecting plant uptake include soil or sediment organic content, carbon exchange capacity, oxide and carbonate content, redox potential, formulation used, and total metal content. In general, mercury uptake in plants could be related to pollution level. With lower levels of mercury pollution, the amounts in crops are below the permissible levels. Aquatic plants have shown to be bioaccumulators of mercury. Mercury concentrations in the plants (stems and leaves) are always greater when the metal is introduced in organic form. In freshwater aquatic vascular plants, differences in uptake rate depend on the species of plant, seasonal growth-rate changes, and the metal ion being absorbed. Some of the mercury emitted from the source into the atmosphere is absorbed by plant leaves and migrates to humus through fallen leaves. Mercury-vapor uptake by leaves of the [C.sub.3] species oats, barley, and wheat is five times great er than that by leaves of the [C.sub.4] species corn, sorghum, and crabgrass. Such differential uptake by [C.sub.3] and [C.sub.4] species is largely attributable to internal resistance to mercury-vapor binding. Airborne mercury thus seems to contribute significantly to the mercury content of crops and thereby to its intake by humans as food. Accumulation, toxicity response, and mercury distribution differ between plants exposed through shoots or through roots, even when internal mercury concentrations in the treated plants are similar. Throughfall and litterfall play a significant role in the cycling and deposition of mercury. The possible causal mechanisms of mercury toxicity are changes in the permeability of the cell membrane, reactions of sulphydryl (--SH) groups with cations, affinity for reacting with phosphate groups and active groups of ADP or ATP, and replacement of essential ions, mainly major cations. In general, inorganic forms are thought to be more available to plants than are organic ones.
Plants can be exposed to mercurials either by direct administration as antifungal agents, mainly to crop plants through seed treatment or foliar spray, or by accident. The end points screened are seed germination, seedling growth, relative growth of roots and shoots, and, in some case, studies of leaf-area index, internode development, and other anatomical characters. Accidental exposures occur through soil, water, and air pollution. The level of toxicity is usually tested under laboratory conditions using a wide range of concentrations and different periods of exposure. Additional parameters include biochemical assays and genetical studies. The absorption of organic and inorganic mercury from soil by plants is low, and there is a barrier to mercury translocation from plant roots to tops. Thus, large increases in mercury levels in soil produce only modest increases in mercury levels in plants by direct uptake from soil. Injuries to cereal seeds caused by organic mercurials has been characterized by abnormal germination and hypertrophy of the roots and coleoptile.
Mercury affects both light and dark reactions of photosynthesis. Substitution of the central atom of chlorophyll, magnesium, by mercury in vivo prevents photosynthetic light harvesting in the affected chlorophyll molecules, resulting in a breakdown of photosynthesis. The reaction varies with light intensity. A concentration and time-dependent protective effect of GSH seems to be mediated by the restricted uptake of the metal involving cytoplasmic protein synthesis. Plant cells contain aquaporins, proteins that facilitate the transport of water, in the vacuolar membrane (tonoplast) and the plasma membrane. Many aquaporins are mercury sensitive, and in AQP1 a mercury-sensitive cysteine residue (Cys-l89) is present adjacent to a conserved Asn-Pro-Ala motif. At low concentrations mercury has a toxic effect on the degrading capabilities of microorganisms. Sensitivity to the metal can be enhanced by a reduction in pH, and tolerance of mercury by microorganisms has been found to be in the order: total population [greater than] nitrogen fixers [greater than] nitrifiers. Numerous experiments have been carried out to study the genetic effects of mercury compounds in experimental test systems using a variety of genetic endpoints. The most noticeable and consistent effect is the induction of c-mitosis through disturbance of the spindle activity, resulting in the formation of polyploid and aneuploid cells and c-tumors. Organomercurials have been reported to be 200 times more potent than inorganic mercury. Exposure to inorganic mercury reduces mitotic index in the root-tip cells and increases the frequency of chromosomal aberrations in degrees directly proportional to the concentrations used and to the duration of exposure. The period of recovery after removal of mercury is inversely related to the concentration and duration of exposure.
Bacterial plasmids encode resistance systems for toxic metal ions, including [Hg.sup.2+] functioning by energy-dependent efflux of toxic ions through ATPases and chemiosmotic cation-proton antiporters. The inducible mercury resistance (mer) operon encodes both a mercuric ion uptake and detoxification enzymes. In gram-negative bacteria a periplasmic protein, MerP, an inner-membrane transport protein, MerT, and a cytoplasmic enzyme, mercuric reductase, the MerA protein, are responsible for the transport of mercuric ions into cells and their reduction to elemental mercury, Hg(II). In Thiobacillus ferrooxidans, an acidophilic chemoautotrophic bacterium sensitive to mercury ions, a group of mercury-resistant strains, which volatilize mercury, has been isolated. The entire coding sequence of the mercury-ion resistance gene has been located in a 2.3 kb fragment of chromosomal DNA (encoding 56,000 and 16,000 molecular-weight proteins) from strain E-15 of Escherichia coli. Higher plants and Schizosaccharomyces pombe respond to heavy-metal stress of mercury by synthesizing phytochelatins (PCs) that act as chelators. The strength of Hg(II) binding to glutathione and phytochelatins follows the order: [gamma]Glu-Cys-Gly[less than][([gamma]Glu-Cys).sub.2]Gly[less than][([gamma]Glu-Cys).sub.3]Gly[less than][([gamma]Glu-Cys).sub.4]Gly. Suspension cultures of haploid tobacco, Nicotiana tabacum, cells were subjected to ethyl methane sulfonate to raise mercury-tolerant plantlets. [HgCl.sub.2]-tolerant variants were selected from nitrosoguanidine (NTG)-treated suspension cell cultures of cow pea, Vigna unguiculata, initiated from hypocotyl callus and incubated with 18 [micro]g/ml [HgCl.sub.2] Experiments have been carried out to develop mercury-tolerant plants of Hordeum vulgare through previous exposure to low doses of mercury and subsequent planting of the next generation in mercury-contaminated soil. Phytoremediation involves the use of plants to extract, detoxify, and/or sequester environmental pollutants from soil and water. Transgenic plants cleave mercury ions from methyl-mercury complexes, reduce mercury ions to the metallic form, take up metallic mercury through their roots, and evolve less toxic elemental mercury. Genetically engineered plants contain modified forms of bacterial genes that break down methyl mercury and reduce mercury ions. The first gene successfully inserted into plants was merA, which codes for a mercuric ion reductase enzyme, reducing ionic mercury to the less toxic elemental form. MerB codes for an organomercurial lyase protein that cleaves mercury ions from highly toxic methyl mercury compounds. Plants with the merB gene have been shown to detoxify methyl mercury in soil and water. Both genes have been successfully expressed in Arabidopsis thaliana, Brassica (mustard), Nicotiana tabacum (tobacco), and Liriodendron tulipifera (tulip poplar). Plants currently being transformed include cattails, wild rice, and Spartina, another wetland plant. The problem of mercury contamination can be reduced appreciably by combining the standard methods of phytoremediation--removal of mercury from polluted areas through scavenger plants--with raising such plants both by routine mutagenesis and by genetic engineering. The different transgenics raised utilizing the two genes merA and merB are very hopeful prospects.
The use of mercury as poison has been known from the beginning of civilization and recorded in ancient oriental and Roman literature (Li, 1948). The medicinal use of mercurials can also be traced back more than 300 years, ranging from treatment for syphilis and various skin disorders (Farler, 1952), to the antiseptic action of [HgCl.sub.2], to use for diuretic and chemotherapeutic purposes (Webb, 1966). The overall awareness of the toxicity of mercury has now stopped the use of mercurials as pharmaceutical agents. However, increasing uses for mercury in modern technology have led to a progressive increase in environmental pollution and consequent widespread mercury poisoning (Kazantzis, 1980). A well-known example is the death in the 1960s of hundreds of people in Central and South Asia through the accidental consumption of seed grains that had been treated with a mercury-based fungicide. The Minamata disaster in Japan involved the conversion of effluents containing elemental mercury to bioavailable methyl m ercury, which entered the aquatic food chain (Bowen, 1979).
Mercury poisoning has again become a problem of current interest as a result of environmental pollution on a global scale. Extensive information is available: a bibliography by Hunter (1978); a discussion of environmental aspects by Hartung and Dinman (1972); a review by D'Itri and D'Itri (1977); and consideration of pharmacological and toxicological aspects by Clarkson (1972), Friberg and Vostal (1972), Miller and Clarkson (1973), Nordberg (1976), and the Swedish Expert Group (1971). Environmental health criteria have been considered by the World Health Organization (1976).
III. Occurrence and Properties of Mercury
Mercury is a rare element, ranking sixteenth from the bottom of the list of elements in abundance in the earth. It is ubiquitous, being present in trace amounts throughout the lithosphere, the hydrosphere, the atmosphere, and the biosphere, as well as in igneous rocks of all classes (Goldwater, 1971). Normal soils typically contain 20--150 ppb mercury, but close to known deposits the level can reach as high as 80% (WHO, 1976). Generally, mercury binds strongly to the organic components of soil, so that mobility by leaching is minimal and contamination of ground water is unlikely unless mercury leaches from a municipal landfill (USEPA, 1984).
The natural source of mercury independent of man's actions occurs as a general cycle (Fig. 1; O'Neill, 1985). It is transported to surface waters by soil erosion and is circulated into the atmosphere by a natural degassing of the earth's crust and oceans. Natural emissions account for two-thirds of the input; manmade releases form about one-third (Berlin, 1986; Burg & Greenwood, 1991; Korringa & Hagel, 1974; NRC, 1978). The annual anthropogenic release of mercury on a global basis was about 3 x [10.sup.6] kg around the year 1900 and increased about three times during the 1970s. A considerable amount is emitted to air (45%); 7%, to water; and 48%, to land.
The most important forms of mercury to which living organisms are exposed can be placed into three broad categories having different pharmacokinetic properties. Of these, elemental mercury is usually referred to as mercury vapor when present in the atmosphere or as metallic mercury in liquid form. It is of considerable importance toxicologically due to relatively high vapor pressure and a certain water (about 20 [micro]g/l) and lipid solubility (5-50 mg/l; Berlin, 1986). Mercury exists in ionic form as [Hg.sup.2+] (mercuric) and [Hg.sup.+] (mercurous). The former readily forms complexes with organic ligands, notably sulphydryl groups. [HgCl.sub.2] is highly soluble in water and highly toxic; [Hg.sub.2][Cl.sub.2] is less soluble and correspondingly less toxic.
In organic mercury compounds, mercury forms a covalent bond with carbon. The principal forms are alkyl mercurials (methyl- and ethyl mercury), aryl mercurials (phenyl mercury), and alkoxy alkyl mercury diuretics. Organic mercury cations form salts with inorganic and organic acids and react readily with biologically important ligands, notably sulphydryl groups. Perhaps, they also pass easily across biological membranes. The major difference among these various organomercury cations is that the stability of carbon-mercury bonds in vivo varies considerably. Thus alkyl mercury compounds are more resistant to biodegradation than are either aryl mercury or alkoxy mercury compounds (Clarkson, 1977).
IV. Uptake and Distribution of Mercury
Mercury has been shown to be released from sediments and accumulated by plants like Ceratophyllum demersum and Anodonta grandis (Hammer et al., 1988). Both gold and mercury have been located in bottom sediments and aquatic plants, aquatic lichens, and dwarf birch and larch, in a river receiving effluents from a plant recovering gold from ores by mercury (Taisaev, 1991). Along the Madeira River, in the Amazon Basin, the highest plant values of mercury, 0.91 and 1.04 [micro]g/g, were obtained in 1986 for Victoria amazonica and Eichornia crassipes, respectively (Martinelli et al., 1988).
A major source of mercury waste comes from mercury cells in the chloralkali industry and from sludge deposited in landfills or discharged into waterways in industrialized countries. A maximum mercury accumulation of 1.5 [micro]g/month was reported near two industrial plants in Harjavatta, Finland, with high levels in moss bags made of Sphagnum girgensohnii (Hynninen & Lodenius, 1986). The level of mercury in the Aveiro Lagoon, Portugal, was high (766 ppm) in sediment and 4 ppm in Convolvulus sp. (De Koe et al., 1988).
Considerable amounts of mercury may be added to agricultural land with fertilizers, lime, and manures (see Andersson, 1979). The use of metal-contaminated sludges, solid wastes, or fertilizers as soil amendments may also cause significant contamination of agricultural soils and crops. The amounts of the metals present in sewage sludges available to the plant depend on such variables as cation-exchange capacity, soil pH, amount of sludge applied, and plant species or variety. Crop plants grown on sludge-amended soils have shown elevated metal concentrations in tissues, with occasional metal toxicity (Page, 1974).
The accumulation of mercury when added to soil was found to decrease after the crop harvest in the order: Oryza sativa [greater than] Brassica oleracea var. capitata [greater than] Brassica rapa [greater than] Zea mays [greater than] Sorghum vulgare [greater than] Triticum aestivum. Permissible levels of soil mercury were 17 mg/kg on calcareous and 6 mg on acid soil (Wang et al., 1982). The critical value of mercury is 0.5 ppm. High pH value, abundant lime, and accumulated salt in soil reduced its uptake by plants(Xuexun & Linhai, 1991).
The most important sources of contaminating agricultural soil have been the use of organic mercurials as a seed-coat dressing to prevent fungal diseases in seeds, of mercury sulphate as a root dip, and of phenyl mercuric acetate (PMA) for the treatment of apple scab (Frank et al., 1976a, 1976b). Mercurials have also been used for to control fungal turfgrass diseases and infestations of crabgrass (Digitaria sp.; MacLean et al., 1973), leading to contamination of golf-course soil (Mathews et al., 1995).
In many countries, seeds are effectively treated before being imported to prevent the introduction of fungi. The main crops that require treatment are wheat and barley, followed by oats, rye, maize, rice, and seed potatoes, as well as a variety of fodder crops. Organomercury compounds are highly effective but directly contaminate the soil, as mercury complexes with the organic humic acid component. In general, the effect of treatment on germination is favorable when recommended dosages are used. Injury to the seed increases in direct proportion to increasing rates of application (Hsi, 1956; Kaohler & Beaver, 1954; Purdy, 1956).
The use of mercury compounds as agricultural seed dressings has resulted in mercury accumulation and toxicity in avian and mammalian seed eaters and in avian predators of these herbivores (Fimreite, 1970; Fimreite et al., 1970; Johnels et al., 1979; Tejning, 1967). Consequently, the use of alkyl mercury compounds as a seed dressing was prohibited in most industrialized countries in the late 1960s.
In Poland the highest mercury contents in plants were recorded in greenhouse vegetables, followed by plants in industrialized areas. Lettuce and parsley leaves had the highest concentrations; tomatoes, potatoes, and cucumbers, the lowest. Fruits and grains bioaccumulated much less mercury than did vegetables. There was no clear correlation between mercury concentrations in the soil and the plants grown on it (Szymczak & Grajeta, 1992).
In soil and plant litter samples from the western part of the Zonien (Soignes) Forest near Brussels, Belgium, mercury concentration was directly related to organic matter content (Muhaya et al., 1993). In most soils, mercury content varies with depth, in particular in virgin soils, related to sampling depth (Andersson, 1979). Organic soils commonly have higher average mercury contents than do mineral soils (Frank et al., 1976a; John et al., 1975). A highly significant correlation exists between mercury and organic-matter content in the top layer of forest soils (Lag & Steinnes, 1978). Total concentrations of mercury in the contaminated soil do not indicate the amount of mercury taken up by plants; for example, in highly contaminated soils, grasses absorb more mercury than legumes (Lucena et al., 1993).
The deposition of mercury as precipitation in forest catchment areas depends on the seasonal atmospheric content of mercury, as observed in Scandinavia (Iverfeldt, 1991). The accumulation of mercury particles in industrialized areas depends on increased trichome frequency and dust-collecting efficiency of five tropical plants studied: Cassia siamea, Calotropis pracera, Ipomoea fistubosa, Zizyphus mauritiana, and Mangifera indica (Rao & Dubey, 1992).
Toxic metal ions are thought to enter cells by means of the same uptake processes that move essential micronutrient metal ions. Mercury, a class B metal, preferentially binds with sulphur-and nitrogen-rich ligands (e.g., amino acids).
The uptake of mercury in terrestrial plants has been the subject of numerous investigations (see Adriano, 1986; Kaiser & Tolg, 1980), mostly in agricultural crops under controlled experimental conditions and almost always with mercury loads far above normal levels. In general, the availability of soil mercury to plants is low, and there is a tendency for mercury to accumulate in roots, indicating that the roots serve as a barrier to mercury uptake (Gracey & Stewart, 1974). The fraction of mercury retained in the roots is about 20 times that observed in the shoots and is closely related to the [NH.sub.4]OAc-extractable mercury in the soils (Lindberg et al., 1979). Mercury concentration in aboveground parts of plants appears to depend largely on foliar uptake of [Hg.sup.0] volatilized from the soil. Mercury compounds applied to some aboveground parts of plants can be readily translocated into others (Adriano, 1986). The mercury content in crop plants grown on soils low in mercury are reported to be in the same range as that in the soils (Gracey & Stewart, 1974). In cereals, the content in grain is about three to ten times lower than that of the straw. Other work has indicated still lower levels of mercury ([sim]1-2 ng/g) in grains of barley and wheat. Even at these very low levels, foliar uptake of [Hg.sup.0] from the air plays a significant role.
The factors affecting the amounts of metal absorbed by a plant are those that control: the concentrations and speciation of the metal in the soil solution; the movement of the metal from the bulk soils to the root surface; the transport of the metal from the root surface into the root; and its translocation from the root to the shoot (Chaney & Giordano, 1977; Wild, 1988). Plant uptake of mobile ions present in the soil solution is largely determined by its total quantity in the soil, but, in the case of strongly adsorbed ions, absorption depends more on the amount of root produced (Wild, 1988). Roots possess a significant cation-exchange capacity, due largely to the presence of carboxyl groups, which may be involved in moving ions through the outer part of the root to the plasmalemma, where active absorption takes place. The uptake of metals from soils is greater in plants grown in pots of soil in the greenhouse than from the same soil in the field (De Vries & Tiller, 1978; Page & Chang, 1978), probably due to differences in microclimate and soil moisture and because the roots of container-grown plants grow solely in contaminated soil, whereas those of field-grown plants may reach down to less-contaminated soil. Translocation within the plant is relatively lower for mercury than for other metals.
Some mercury accumulation has been observed in mushrooms, aquatic plants, carrots, and potatoes. Aquatic Posidonia oceanica could be a biological indicator for mercury in sediments (Ferrara et al., 1989). Mercury concentrations in spruce needles increased continuously with age (Wyttenbach et al., 1989). Lichens and mosses may concentrate mercury on their surfaces (see Merian, 1991). Plant-specific uptake of mercury has been studied in bryophytes, lichens, wetland plants, woody plants, and crop plants. Factors affecting plant uptake include soil or sediment organic content, cation-exchange capacity, oxide and carbonate content, redox potential, and total metal content (Crowder, 1991).
Among higher plants, degree of mercury toxicity varies with the formulation used and with the plant species in addition to the concentration of the chemical. In general, mercury uptake in plants may be related to the level of pollution. Sixteen vegetable crops grown on a garden plot, exclusively treated with residential compost for six years, had methyl mercury levels averaging 12.8% of the total edible tissue mercury content (Cappon, 1987).
Exposure to different levels of 2-methoxy ethyl mercury chloride (Aretan) and [HgCl.sub.2] do not affect dry matter and grain yields of Triticum aestivum but prevent germination of Phaseolus vulgaris. Translocation to grain is low (Semu et al., 1985). In Lycopersicon esculentum, uptake of mercury is directly related to the level of mercury pollution (Zhou & Zhao, 1992).
In Swiss chard (Beta vulgaris), grown in compost-amended soil, the DTPA-extractable and total metal contents are highly correlated, but not with plant metal concentrations or metal uptake (Warman et al., 1995). The mercury content of potato tubers is not affected by fertilizers or irrigation (Blaskova, 1994).
The uptake of mercury from soil following long-term application of wastewater in Tehran, Iran, shows elevated levels in the upper layers of soil but relatively low levels in the vegetables (Shariatpanahi & Anderson, 1986). Comparison of the heavy-metal contents of legume seeds of different origin and variety indicates a complex pattern of environmental and genetic factors that contribute to the specific metal contents of individual harvests. At the level of varieties, environmental factors seem to exhibit a more important influence on the specific accumulation of heavy metals than do genetic factors. In contrast, on the level of species or genera, the accumulation of heavy metals seems to be dominated by genetic factors (Gross et al., 1987).
Widespread studies have shown that with lower levels of mercury pollution, the amounts in crops are below the permissible levels. In a series of sampling of food crops and fruits from all over the world, including Czechoslovakia, Taiwan, and South India, with soil contaminated by mercury through irrigation or fertilizers, the amount of mercury present in the crops was below the daily tolerable intake recommended by WHO (Dembinska et al., 1994; Ellen et al., 1990; Lin et al., 1992; Ruales & Nair, 1993; Srikumar, 1993).
During a soil bioassay using the rapid-cycling Brassica rapa selections, in a sand soil, bloom initiation was slowed by [less than]10mg Hg/kg. Lettuce (Lactuca sativa) emergence was less sensitive but survival of Daphnia magna more sensitive to mercury. A similar relationship among the bioassays was observed for two finer-textured soils at soil metal concentrations of [greater than]200 mg/kg. Enzyme assays were not sensitive to mercury contamination (Sheppard et al., 1993).
Analysis of about 2,500 vegetable samples and about 650 soil samples showed that a considerable part of the vegetables, especially those with edible roots, contained mercury usually on the order of thousandths of milligrams per kilogram (Zawadzka et al., 1990). Similar results were obtained in cultivars of strawberry and raspberry, collected from five regions (Surdel, 1991).
The uptake and excretion of mercury have been studied in seedlings of woody plants (Kotov, 1983), in plant shoot of oats and lettuces (Staiger, 1983), in the eelgrass Zostera marina (Lyngby & Brix, 1982), in crops grown on sludge-treated soil (Davis, 1984), and in spruce (Picea abies) seedlings (Godbold & Huettermann, 1985). Plant growth was reduced in all soils containing 50 mg Hg/kg, and the plants contained 1.4-37.6, 0.4-1.2, and 80-800 mg Hg/kg in leaves, stems, and roots, respectively, making them potentially hazardous to grazing animals (Weaver et al., 1984).
In plant species around a chloralkali factory in India, accumulation in leaves was the highest, followed by the stem and the root. A significant correlation was noted between the mercury concentration of the soil and the plant tissues and between different tissues (Lenka et al., 1992; Shaw & Panigrahi, 1986). Amounts of mercury in the plant tissues of Lolium sp. (ryegrass; Singh & Jeng, 1993) and Rosmarinus officinalis L. (rosemary) were related to its concentration in the soil (Barghigiani & Ristori, 1995).
Samples of six common plant species collected in old mining areas near Prince George, British Columbia and Mount Amiata, Tuscany, show remarkable similarities in the variation of plant:soil mercury-concentration ratio, with soil mercury content irrespective of species or other biological differences. In contrast, plants sampled in geothermally active areas of New Zealand, Hawaii, and around Mount Saint Helens, in Washington State, exhibit more individuality in the concentration ratio to soil-mercury relationship. Thus, specific local environmental factors strongly influence the accumulation of mercury in plants even when the immediate soil concentrations are the same (Siegel et al., 1987).
Among aquatic plants, in a natural sediment enriched with [CH.sub.3]HgCl (4 mg Hg/kg fresh weight), Elodea densa showed a high accumulation of mercury in leaves, stems, and roots. In long-term experiments, root absorption was the dominating factor of mercury accumulation, the leaves being the principal organ for storage. Two mechanisms are superimposed on this direct transfer: contamination by water, linked to the release of mercury at the water--sediment interface during the initial exposure phase (4 days), and interplant transfers resulting from decontamination processes. Temperature and photoperiod had a strong influence (MauryBrachet et al., 1990).
In seedlings of Picea abies (L.) Karst., root elongation was inhibited by mercury. Mercury reduced potassium, manganese, and magnesium in roots and accumulated iron in root tips. Uptake of [Ca.sup.45] into the roots was increased strongly by [HgCl.sub.2] but only slightly by [CH.sub.3]HgCl (God-bold, 1991). In black spruce from boreal forests in Quebec, sampled in 1990, the difference in tree-ring mercury content associated with the geographical orientation of the discs indicates that daily exposure to sunlight as well as temperature may affect the uptake of mercury and that the mercury observed in the tree rings must have been deposited from the atmosphere onto the tree surface (Zhang et al., 1995). Seasonal changes were recorded in the content of mercury in leaves of Vaccinium myrtillus growing in forests in Espoo, Finland (Koski et al., 1988).
In the cyanobacterium Nostoc calcicola Breb, the [Hg.sup.2+] uptake pattern comprised two distinct phases: rapid binding of the cation to the negatively charged cell surface (first 10 minutes), and its subsequent metabolism-dependent intracellular import. The influx of [Hg.sup.2+] depended to a major extent on photosynthetically generated energy. The significant lowering in the rate of [Hg.sup.2+] uptake, as well as the total cellular [Hg.sup.2+] in the presence of p-chloromercuribenzoate (pCMB), sodium azide ([NaN.sub.3]), N,N'-dicyclohexycarbodimide (DCCD), and thiol (mercaptoethanol), indicates the role of membrane potential, --SH groups, and ATP hydrolysis in regulating [Hg.sup.2+] transport. Although [Cu.sup.2+] antagonized [Hg.sup.2+] intake, [Ni.sup.2+] showed synergism (Pandey & Singh, 1993). In 37 samples of Lentinus edodes (= Lentinula edodes) cultivated on logs and 19 on sawdust substrate beds, mercury contents were significantly higher in the former (Aoyagi et al., 1993; Koio & Lodenius, 1989). I n the epiphytic lichen Hypogymnia physodes and in spruce (Picea abies) needles at 12 sites around smelters in Rudnany and Krompachy, Slovakia, concentration of mercury was generally higher in H. physodes than in the spruce needles. Concentrations of mercury in lichens in Slovakia were relatively high (Lackovicova et al., 1994).
Near a geothermal power plant in southern Tuscany (Italy), high concentrations of mercury occur in mosses, the uptake being unrelated to the species except for Bryum torquescens. Mercury was lower in the soil than in mosses (Baldi, 1988). Young thallus tissues of the epiphytic lichen Parmelia sulcata and surface soil associated with its host trees were collected on Mount Amiata and Mount Etna, Italy. Parmelia from Mount Amiata accumulated mercury from soil degassing, but that from Mount Etna did not. It appears that widely separated populations of the same species can display biogeochemical differences that are best explained on an ecophysiological basis (Bargagli & Markert, 1993).
The mercury contents of vegetables in the Boashen district of Shanghai were found to be high, in the order: leafy vegetables [greater than] root tubers [greater than] fruit vegetables (Feng et al., 1993).
Aquatic plants are shown to be bioaccumulators of mercury. In aquatic macrophytes growing in water bodies around Wroclaw, Poland, partly affected by
atmospheric pollution, effluents of chemical factories, and groundwater contaminated by slag dumps, the highest concentrations of mercury surpass the average values established for background reference sites. The liverwort Scapania undulata originating from a clean, forested mountain stream and cultivated in solutions containing 70-100% sewage from a chemical factory demonstrated an increase in mercury content (40 times in 100% sewage and 20 times in 70% sewage; Samecka & Kempers, 1996).
Four rooted macrophytes (Elodea densa, Ludwigia natans, Lysimachia nummularia, and Hygrophila onogaria) were used to quantify and compare mercury bioaccumulation capacity. Results showed very great accumulation differences after 18 or 21 days of exposure: Mercury concentrations in the plants (stems plus leaves) were always greater when the metal was introduced in organic form, and differences were even greater when initial contamination was via the sediment; bioaccumulation from the water source was about 10 times greater than was that from the sediment; and major interspecies differences emerged in mercury burdens accumulated by the plants, with differences being very small when results were expressed as concentrations, thus taking account of the different biomasses of the species (Ribeyre & Boudou, 1994).
The concentrations of mercury in 13 genera of seaweeds in Thailand ranged from 0-0.839 ng/g dry weight. The brown Fadina tetrastromatica and Dictyota dichotoma and the red seaweed Acanthophora spicifera were the most reliable indicators (Khanianapai et al., 1984).
In freshwater aquatic vascular plants, differences in the rate of uptake depended on the species of plant, seasonal changes in growth rates, and the metal ion being absorbed. Higher concentration of mercury in the plants indicated a proportional increase in the mercury level in the water (Mortimer, 1985). Accumulation of mercury by Azolla was observed to affect growth (Mishra et al., 1987).
Accumulation of mercury was maximum in Hydrilla verticillata, followed by Oedogonium areolatum and Eichhornia crassipes. Accumulation in roots of Eichhornia was about twice as high as that in shoots (Jana, 1988). The level of mercury in Eichhornia crassipes after one week of exposure to 25, 50, and 75 ppm was about 70-75%. The concentration of total free amino acids and the enzymes alanine aminotransferase and aspartate aminotransferase increased with increases in the concentration of mercury (Hussain & Jamil, 1990). Under field conditions, absorption of mercury increased with increasing levels in the culture solution (Tabbada et al., 1989). Such higher accumulation of mercury in roots was also seen in Pistia stratiotes from Nigeria (Sridhar, 1988) and in Lolium perenne (Al-Attar et al., 1988).
In the intertidal freshwater marsh of Quebec City, there was 11.2 times more mercury in plants than in sediment. Scirpus americanus contained more mercury than did other species (Gilbert, 1990). The pH is a major factor in the uptake (Lodenius & Maim, 1990).
Roots of the submerged isoetid aquatic macrophyte Eriocaulon septangulare, cultured for 31 days in sediment contaminated with nontoxic concentrations of inorganic mercury, had significantly higher concentrations and significantly greater mercury content than did plants cultured without added mercury. There was no evidence of the transport of mercury from root to shoot within the plant, although there may have been some transport in the opposite direction (Coquery & Welbourn, 1994).
Water hyacinth (Eichhornia crassipes), common cattail (Typha latifolia), burr reed (Sparganium minimum), and Menyanthes trifoliata roots readily absorb mercury ions from aqueous solutions. The hydrophilic parts of the roots accumulated significantly more mercury than did the hydrophobic parts (Robichaud et al., 1995).
Some of the mercury emitted from the source into the atmosphere is absorbed by plant leaves and migrates to humus through fallen leaves. Consequently, plant leaves can be used as an indicator for the evaluation of mercury currently present in air. Humus is useful for the evaluation of mercury contamination through the air from the past to present (Tamura et al., 1985). A plant life-cycle bioassay for mercury was developed for contaminated soil for comparison with other bioassays (Sheppard et al., 1993).
During a detailed survey of 400 samples, the concentration of mercury in coniferous needles did not vary significantly over eight weeks in summer but did vary significantly between first- and second-year growth. In all tree species, mercury concentrations in needles and leaves were two to three times as high as that in twig tissue from the same branch. Differences in mercury content between tissues of different types and ages constituted a major source of within-site variation in plants of the same species (Rasmussen et al., 1991).
Comparison of six gramineous plant species showed that mercury-vapor uptake by leaves of the [C.sub.3] species Avena byzanlina, Hordeum vulgare, and Triticum aestivum was five times greater than that by leaves of the [C.sub.4] species corn, sorghum, and crabgrass. Such differential uptake by [C.sub.3] and [C.sub.4] species was largely attributable to internal resistance to mercury-vapor binding (Browne & Fang, 1983).
In eastern Denmark, atmospheric mercury contamination of crops contributed more than 90% of the total plant mercury in the green parts, being absorbed into and transported throughout the whole plant. Even in the subterranean part, the atmospheric contribution was approximately 50%. Airborne mercury thus seems to contribute significantly to the mercury content of crops and thereby to intake by humans as food (Mosbaek et al., 1988).
In the acid-precipitation area of suburban Chongqing, China, the average soil mercury rose from 0.158 mg/kg in 1984 to 0,20 mg/kg in 1989, and the mercury content of crops grown on these soils also increased. Both soil and vegetable mercury came mainly from power-plant emissions (Mou & Qing, 1995).
Experiments were conducted to study the absorption, phytotoxicity, and internal distribution of mercury in tobacco plants exposed to elemental mercury vapor ([Hg.sup.0]) through the shoot or to ionic mercury ([Hg.sup.2+]) through the root. Accumulation, toxicity response, and mercury distribution differed between the two exposure routes, even when internal mercury concentrations in the treated plants were similar. Plant exposed to [Hg.sup.0] accumulated mercury in the shoots with no movement to roots. Root-exposed plants showed the accumulation of mercury with movement to the shoots by the tenth day. Inhibition of root and shoot growth occurred at 1.0 [micro]g/ml and above, with very limited tissue damage at higher levels of treatment (Suszcynsky & Shann, 1995).
D. OTHER MODES
Throughfall and litterfall have been shown to play a significant role in the cycling and deposition of mercury in the watershed of Lake Champlain (Rea et al., 1996).
Temporal changes in the mercury content of balsam fir (Abies balsamea) needles and white spruce (Picea glauca) needles, monitored at a site in the southern Canadian shield, indicated a significant increase in the mercury content of needles over the course of a growing season and from one year to the next. Temporal variation is thus a potential source of error when mapping the spatial variation of mercury concentrations in vegetation (Rasmussen et al., 1995).
Experimental data on the accumulation and release kinetics of azalea (Rhododendron sp.) leaves exposed to a constant vapor level of mercury indicate that accumulation of mercury appears to be irreversible, probably as a consequence of chemical transformation (Gaggi et al., 1991). Mercury concentration was determined from the foliage of forests in areas with different levels of pollution in Slovakia. The ranges for two-year-old needles of Picea abies were (in mg/kg): 1.249-4.402 (Rudnany iron ore mines), 0.013-0.749 (nine other industrial regions), 0.021-0.737 (four mountain forests), and 0.053-0.538 (the military area). The moss Pleurozium schreberi contained 3.8-9.1 mg/kg (Mankovska, 1996).
The mean value for mercury content of approximately 100 species of tropical plants sampled on Hainan Island was 0.015 ppm. The mercury levels in conifers, deciduous trees, and herbaceous plants were 0.037, 0.028, and 0.017 ppm, respectively. Leaves had higher levels of the metals than did other plant parts (Wang et al., 1985).
In samples of the epiphytic lichen Hypogymnia physodes, collected from the bark of stems of pine (Pin us sylvestris) , birch (Betula pubescens), and spruce (Picea abies) at different distances from a wood pulp (sulphide-cellulose) and paper plant in Mantta, southern Finland, mercury levels were slightly increased (Kytomaa et al., 1995). The highest background values were found in western and southern Finland, which may indicate an influx of mercury with the prevailing winds. Very high concentrations (up to 36 ppm) were found near chloralkali works. No correlation was found between mercury concentrations and population density, annual precipitation, or the mercury content of soil and groundwater (Lodenius, 1981).
V. Toxic Effects of Mercury on Plants
Studies of micronutrient requirements and toxic effects of trace metals on soil organisms and native plants in fields are limited. Usually the effects of metals are examined in sterile and much-simplified laboratory conditions, which may differ from field conditions in different degrees (Ross, 1994). Much research has been directed at the effects of metals on food-plant production but, until recently, rather less on trace-metal cycling in natural ecosystems.
The two categories of toxic effects defined are acute toxicity--a large dose of the chemical for short duration, which is usually lethal--and chronic toxicity--a low dose over a long period of time, which can be lethal or sublethal (Alderdice, 1967). The three main aspects of study are biomagnification, or the transfer and accumulation of metals up to the food chain; developing more useful, relevant, and statistically acceptable criteria for selection of metal toxicity thresholds in soil; and empirical study of the effects of other metal toxicity that may modify the thresholds for individual metals. The main problems are that phytotoxicity thresholds differ with plant species, that soil properties influence the rates at which metals transfer to plants, that roots may sequester metals and prevent or reduce translocation to the leaves, that no chemical or toxicant interactions are taken into account, and that changes in foliar chemistry may be influenced by other environmental factors, such as the availability , pH, redox, or salinity of water.
At the cellular level, the possible mechanisms by which toxic metals may effect damage include blocking functional groups of biologically important molecules, such as enzymes, polynucleotides, or transport systems for nutrient ions, displacing and/or substituting essential metal ions from biomolecules and functional cellular units, denaturing and inactivating enzymes, and disrupting cell and organelle membrane integrity (Ochiai, 1987). The possible causal mechanisms of mercury toxicity are changes in the permeability of the cell membrane, reactions of sulphydryl (-SH) groups with cations, affinity for reacting with phosphate groups and active groups of ADP or ATP, and replacement of essential ions, mainly major cations (Kabata-Pendias & Pendias, 1984). In general, inorganic forms are thought to be more available to plants than are organic ones (Linster, 1991).
Plants can be exposed to mercurials either by direct administration as antifungal agents, mainly to crop plants through seed treatment or foliar spray, or by accident. In the former, a wide variety of antifungal mercurials are involved, mainly organic compounds and combination of several compounds. Seed treatment is the more accepted method. A large amount of data has accumulated in this aspect, and the end points screened are seed germination, seedling growth, relative growth of root and shoot, and, in some case, studies of leaf-area index, internode development, and other anatomical characters.
Accidental exposure comes through soil, water, or air pollution. The level of toxicity is usually tested under laboratory conditions using a wide range of concentrations and different periods of exposure. The plants tested are mainly aquatic plants for water pollution and local species available at polluted regions under different soil and altitude conditions. These studies include screening for mercury toxicity in areas following contamination with sewage sludge or factory effluents. In both sets of experiments the parameters screened are similar. Additional parameters include biochemical assays and genetical studies.
The greatest amount of information available in on seed germination and the growth of different plant species in fields that have been exposed to mercurials, given as fungicides. Other aspects include laboratory experiments to test the relative efficacy of mercury compounds against fungal cultures, effects on plant (shoot plus root) growth, and cell cultures.
A. HIGHER PLANTS
The absorption of organic and inorganic mercury from soil by plants is low, and there is a barrier to mercury translocation from plant roots to tops. Thus large increases in soil mercury levels produce only modest increases in plant mercury levels by direct uptake from soil. Mercury salts in soil may be reduced by biological and chemical reactions to mercury metal or methylated compounds, which may volatilize and be taken up through the leaves, a much more efficient process than via the roots. This is important for plants grown in enclosed spaces, such as greenhouses. Residues of mercury pesticide or fungicide sprays are, in some cases, taken up by plants (e.g., Oryza sativa) and translocated to edible portions. Phenyl-Hg salts have been shown to hasten plant senescence and inhibit photosynthesis. Mercurial solutions used as seed treatments may reduce seed viability if the mercury concentration is too high or if the seed is stored with too high a moisture content (Anonymous, 1979).
1. Seed Germination, Growth, and Development
Phytotoxic effects of mercury compounds have been reported in several plants, including Triticum aestivum (Mosso, 1979), Oryza sativa (Shimizu et al., 1975), and several other grain crops (Blomqvist, 1985; Karlberg, 1976). In general, the degree of impact depends on the concentration, the formulation, the mode of application, and the cultivar.
The seed injury caused by organic mercurials to cereals has been characterized by abnormal germination. The effect is a characteristic hypertrophy of the roots and coleoptile of cereal seedlings, where higher dosages of fungicides are used or when storage conditions are faulty (Crosier & Keitt, 1934; Noll, 1938; Purdy, 1956; Weston & Booer, 1935). The primordia undergo extensive thickening and develop irregular carnations and lobes, and in the apical meristem of the plumule, cell division is inhibited and extreme enlargement of the existing cells takes place (Sass, 1937).
The primary effect of mercury may possibly be on the embryo itself, and effects on the endosperm are of secondary importance. Mercury strongly interferes with the -SH system in living cells by causing the formation of -S-Hg-S- bridge. Such a breakdown of the normal -SH system may affect both germination and subsequent growth of the young embryo, because these tissues are particularly rich in -SH groups.
In Oryza sativa, elongation of seedlings was inhibited at a concentration of 5 x [10.sup.-4] M [HgCl.sub.2] and germination stopped at 6 x [10.sup.-3] M. Root growth was more affected than was shoot growth. Inhibition was reversed with organic acids, cations, ethylene diamine tetra acetic acid, and hormones. Marked inhibition of a-amylase resulted with [HgCl.sub.2] treatment, which could be prevented by gibberellic acid and cAMP. The response was cultivar specific. Mercury also inhibited significantly the mobilization of total nitrogen and phosphate reserves from grains during seedling growth (Varshney, 1991). Of different formulations used in seed treatment of Oryza sativa, emisan (2-methoxy ethyl mercury chloride, 0.01%) + streptocycline (0.01) was the most effective for control of seed rot and resulted in maximum germination (Singh et al., 1992). However, emisan was highly toxic when applied following heavy rains, significantly reducing germination and seedling vigor (Sharma & Sharma, 1993). In seeds of O ryza sativa cv. Al-Ahssa and Medicago sativa cv. Al-Ahssa exposed to [HgCl.sub.2] the percentages of germination and seedling growth decreased with increasing concentrations, the effects being more on root growth (Al-Helal, 1995).
Differential changes were induced by 0.5-2.0 mM [HgCl.sub.2] on the root and stern structure of Triticum aestivum cv. WL 711 and anatomical changes at high concentrations were drastic. Mercury induced the secretion of a mucilaginous substance on the epidermal surface and suppressed the differentiation of roots. In stems, the diameter, number of vascular bundles, and cell sizes decreased, and cell-wall thickness in epidermal and hypodermal tissues increased (Setia et al., 1994). The damage was not recorded with recommended practices (Singh & Ghosh, 1991). In Triticum aestivum CV. Pak-81, mercury inhibited germination, as well as development and chlorophyll contents, retarded root-shoot length, and decreased root-shoot dry weights of seedlings and soluble protein content of the shoot (Iqbal & Majeed, 1991). Two peroxidase isozyme bands were observed in seedlings (Zhang et al., 1989).
Retardation of the growth of seedlings from small seeds was ameliorated by fungicide application. Mechanical stress during sowing was reduced in large seeds or seeds treated with fungicide plus PGR plus fertilizer in Germany (Heyland & Meer, 1992). Seed treatment with 150 g triadimenol reduced the length of subcrown internodes and inhibited tillering in winter wheat varieties grown at Long Ashton, United Kingdom. The reduction in subcrown internode length may improve tolerance of freezing soil temperatures, and the inhibition of excessive tillering may lead to repartitioning of assimilates into grain (Anderson, 1989).
Mercuric chloride, applied in droplets on the primary roots of Zea mays, reduced their elongation and inhibited their graviresponse (Pilet & Versel, 1981). Soaking seeds for 24 hours decreased germination, seedling growth, and protein and chlorophyll contents and increased carotenoid content (Kalimuthu & Sivasubramanian, 1990).
Foliar application of 10 ppm PMA (antitranspirant) 60 days after sowing to Hordeum vulgare cv. RD 31 increased the grain yield (Maliwal et al., 1993). The effects of higher doses, however, on Hordeum vulgare and Triticum aestivum cultivars included reduction in the number of well-germinated grains (Benada, 1992).
In Pennisetum typhoideum (P. americanum) cv. PHB-14, Medicago sativa cv. Raska and Abelmoschus esculentus cv. Pusa savni, visible injury to leaves increased and chlorophyll content and total plant dry matter decreased with exposure to increasing concentrations of mercury (Mhatre & Chaphekar, 1984).
Exposure to mercurial fungicides reduced infection by pathogens in green gram ( Vigna radiata), black gram ( Vigna mungo), and pigeon peas (Cajanus cajan) but also reduced dry-matter yields by 95%, 98%, and 91% by mercury (Kala et al., 1992). Of five mercury-based fungicides, Agrosan GN, mancozeb, thiram, and thiram plus captan were most effective, with less than 10% seed infection and improved germination of Zea mays seed (Paul & Mishra, 1994).
Inoculation of Vigna radiata V. mungo, and Arachis hypogaea seeds with Rhizobium stains and treatment with emisan or bavistin (carbendazim) significantly reduced Rhizobium population and nodulation (Ghosh, 1995). Treatment of seeds of two Phaseolus aureus [Vigna radiata] cultivars with higher concentrations of mercuric acetate inhibited germination, hypocotyl length, mobilization of total nitrogen from cotyledons to seedlings, and protease activity in seeds during germination (Varshney, 1990). With increasing concentrations of [HgCl.sub.2] the respiration rates of seedlings declined, as did the levels of total nitrogen, total sugar, DNA, and RNA in embryos with concomitant accumulation in cotyledons. Gel electro-phoretic studies revealed major disruption and increase in number of protein bands (Nag et al., 1989).
High concentrations of [HgCl.sub.2] reduced seed germination in Phaseolus vulgaris and Brassica campestris, but very low concentrations somewhat increased the rate (Gupta, 1991). The effect of mercury ions with three constant temperatures (T) on Phaseolus vulgaris cv. Contender was studied with respect to shoot and root length, dry matter, chlorophyll (Chl) content and Chl stability index (CSI), contents of soluble (SS) and hydrolysable (HS) sugars, soluble proteins (SP), and total free amino acids (AA). Statistical analysis of the data indicated that T was dominant in affecting CSIa, shoot AA, and root SS, that mercury had a predominant effect on growth parameters and ChI content, and that interaction (T x Hg) was dominant in affecting CSIb, shoot SP, and root HS (Gadallah, 1994). Similar effects were seen with jute, Corchorus olitorius cv. JRO 524 and C. capsularis cv. JRC 321 (Dalal & Bairagi, 1985). In sugarbeet cv. amethyst seeds, effects of mercurials in reducing fungi and affects on germination were r elated to the fungicide and the concentration of mercury (Durrant & Mash, 1992). On Arachis hypogaea, the results were varied (Amaregouda et al., 1994).
The effect of mercury on root length, stem length, leaf area, and dry-matter production of Abelmoschus esculentus is inversely proportional to the concentration (Ganesan & Manoharan, 1983). PMA applied to both sides of sunflower leaves caused a greater closure of the stomata on the upper side but increased cuticular resistance to gas diffusion only in leaves of plants growing in dry soil, suggesting that soil-moisture stress as well as PMA decreases the permeability of nonstomatal epidermal cells (Moreshet, 1975).
In Allium cepa, Amaranthus sp., Beta vulgaris, Brassica oleracea L. var. capitata, Chinese cabbage, Coleus blumei, Cucumis sativus, Hibiscus esculentus, Pisum sativum, Raphanus sativus, and Lycopersicon esculentum exposed to 14 fg Hg vapor/l, radicle emergence was not substantially reduced, but in Mentha sylvestris and Lactuca saliva it was reduced by 50%. Allium cepa, Lactuca sativa, and Pisum sativum showed reduction in the growth of both seedling roots and shoots. Others showed inhibition in either shoot or root growth, while in some there was no inhibition and occasionally some stimulation of growth (Siegel et al., 1984).
Pollen germination and tube growth of Lilium longiflorum were affected by concentrations of 3-100 [micro]M of chlorides of [Hg.sup.2+] The main effect was abnormal cell-wall organization (Sawidis & Reiss, 1995). In field trials, foliar administration to Sorghum vulgare cv. CSH 5 of different formulations of phenyl mercury acetate did not affect grain or fodder yields and yield components (Kaore et al., 1993).
Application of dried sewage sludge and a superabsorbent hydrogel (Evergreen 500) in the first year significantly increased the growth of apple seedlings in perforated bags (Awad et al., 1995). In long-term experiments, high application rates of sewage sludge and pig slurry were tolerated by silage maize and grass. The mercury content was influenced only by the excessive application of sewage sludge (Siegenthaler & Stauffer, 1991). In Styria, Austria, both silage maize and grass showed better growth after sewage-sludge application, with no significant negative effects on soils or plants (Hyll & Nestroy, 1993).
In Stylosanthes guianensis seeds, soaked for five minutes in organic mercurials, the length of root cells in seedlings decreased with increasingly negative water potential and with mercury concentration. A possible effect of mercury is suggested to promote ethylene production, which would inhibit root growth (Delachiave et al., 1990). In cultivars of Saccharum officinarum, lower concentrations of organic mercurial fungicides improved sprouting (Singh & Bains, 1992) and gave higher cane and sugar yields (Ali & Srivastava, 1995). Other formulations, like 0.25% emisan, were not effective (Bhale & Hunsigi, 1994). Soil mercury and organic matters affected development and yield of summer millet (Zhang & Lin, 1992).
Three aquatic plants, Hydrilla verticillata, Pistia stratiotes, and Salvinia molesta, treated with different amounts of mercury were severely affected. Foliar injury, chlorophyll content, and phytomass showed perceptible effects with increasing exposure. In floating plants, a positive relation was obtained between Leaf Injury Index (LII) and concentration (Mhatre & Chaphekar, 1985). Bioaccumulation led to physiological changes in Hydrilla verticillata (Gupta & Chandra, 1996).
In seeds of Miscanthus floridulus, collected from seven sites (1,600-2,000 m altitude) and of M transmorrisonensis collected from one site (2,600 m) in Taiwan, exposure to mercury reduced the percentage of germination and seedling growth. A 1,000 ppm mercury solution increased the leaching of potassium from seeds. Germination and seedling growth in M. transmorrisonensis were less than in M. floridulus (Hsu, 1993; Hsu & Chou, 1992).
Successive application of mercury and chloride ions stimulated seed germination in Pinus halepensis Mill. (Thalouarn, 1976). Mercury also affected the germination and root growth of Sinapsis alba seeds (Fargasova, 1994). Changes in the pigment content of mulberry plants (Morus alba) exposed to solid waste from a chloralkali facility included the initial appearance of brown patches followed by total browning and fall of all leaves in the exposed plant. Drastic but significant decline in pigment content in exposed plants was observed at higher concentrations of the solid waste and at prolonged periods of exposure (Mohapatra et al., 1990).
During the normal growth cycle of peanut (Arachis hypogaea) cells, cultured in suspension medium, cell aggregates of [less than]0.5 mm were formed during the log phase and grew to aggregates of [greater than]0.5 mm during the late growth phase. Mercury, at low concentrations, allowed continued growth of [greater than]0.5 but not [less than]0.5 populations but arrested growth at high concentrations (1 mM). At low levels (10 [micro]M) and in the presence of 3 mM calcium they had a synergistic effect (Xu & Van-Huystee, 1993). Similar effects of mercury(II) species were seen on cell suspension cultures of Catharanthus roseus (Zhu & Cullen, 1994).
The growth of secondary callus tissue cultures of Ruta graveolens was affected by exposure to mercury in direct proportion to concentrations between [10.sup.-5] and [10.sup.-7] M. The dry weight increased in inverse ratio to the inhibition of fresh weight, due to dehydration of the cells (Maroti & Bognar, 1989).
Cultures of Daucus carota, Ca-68-l0, and Lactuca sativa, Le-67, grown at increasing concentrations of methyl mercury (MeHg), interacted with light synergistically in the expression of MeHg toxicity. Demethylation patterns increased or decreased depending on the species, the concentration of 2,4-D in the medium, and the concentration of methyl mercury. Lactuca sativa was more sensitive than Daucus carota. In this cell population, MeHg toxicity appears to be partly a hormone-mediated and light-sensitive event (Czuba, 1987). Mercury effects in plant senescence were auxin and carbon dioxide sensitive (Spitel & Siegel, 1975).
2. Biochemical Effects
Mercuric cations have a high affinity for sulphydryl (-SH). Because almost all proteins contain sulphydryl groups or disulphide bridges, mercurials can disturb almost any function in which critical or nonprotected proteins are involved (Clarkson, 1972). A mercury ion may bind to two sites of a protein molecule without deforming the chain, or it may bind two neighboring chains together, or a sufficiently high concentration of mercury may lead to protein precipitation. With organomercurials, the mercury atom still retains a free valency electron, so that salts of such compounds form a monovalent ion.
Mercury compounds can bind to the RNA of tobacco mosaic viruses (Katz & Santilli, 1962), several synthetic polyribosomes, and yeast soluble RNA (Kawade, 1963). More rapid and drastic changes occur after inorganic mercury poisoning, perhaps owing to the fact that inorganic mercury binding to nucleosides is almost 10 times stronger than that of methyl mercury (Simpson, 1964). However, RNA content "rebounds" after prolonged exposure to inorganic mercury, and animals can recover from the neurological disturbances (see Merian, 1991).
Mercury affects both light and dark reactions in photosynthesis. The extent of toxicity and the mechanism influencing the photosynthetic apparatus depend largely on the way these phenomena are investigated--that is, in vitro or in vivo experiments--as well as on the age of the plants used (Krupa & Baszynski, 1995).
Mercury strongly inhibited electron transport activity, oxygen evolution, and quenching of chlorophyll fluorescence in photosystem II (PS II) preparations from Hordeum vulgare. Only chloride salts, including NaCl, TMACl, [CaCl.sub.2], and [MnCl.sub.2], could reverse the inhibition by mercury. Chlorine-related sites in PS II were altered by mercury inhibition, suggesting that mercury exerts its action on the donor side of PS II (Bernier et al., 1992; Lee et al., 1992).
PMA inhibits both Hill activity and photophosphorylation (Siegenlthaler & Packer, 1965), as well as the dark reduction of cytochrome [C.sub.554] in whole cells of Chlamydomonas reinhardi, and inhibits either ferredoxin or ferredoxin-NADP oxidoreductase (Hiyama et al., 1970). PMA inhibition of PS II appears to be selective, blocking a site between the 3-(3,4-dichlorophenyl)--1, 1-dimethyl urea-sensitive site and the site inactivated by high concentration of tris buffer (Honeycutt & Krogmann, 1972).
Substitution of the central atom of chlorophyll, magnesium, by mercury in vivo is an important damage mechanism, because it prevents photosynthetic light harvesting in the affected chlorophyll molecules and results in the 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, including mercury. Heavy-metal chlorophylls are formed, some of which are much more stable toward irradiation than is magnesium-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 chlorophyll is inaccessible to heavy-metal ions (Kupper et al., 1996). In Hordeum vulgare Photosystem II submembrane fractions, oxygen evolution was strongly inhibited and chlorophyll fluorescence was severely quenched by mercury. Chloride reversed the inhibitory effect, but calcium did not. It was concluded that mercury perturb s chloride binding and/or functioning (Bemier et al., 1993).
In Pistia stratiotes L., at 20 ppm and above, Hg(II) promoted senescence by decreasing chlorophyll, protein, RNA, dry weight, and activities of catalase and protease as well as by increasing free amino acid content, peroxidase activity, and the ratio of acid to alkaline pyrophosphatase activity over control values (De et al., 1985). Application of mercury in small amounts affected functioning of the donor site of duckweed (Spirodela polythiza) photosystem II through the inhibition of catalase activity in the water-photolysis system. It is suggested that due to the effect of mercury on the rate of electron transport on the donor side of PS II, the ATP:NADPH ratio was changed, leading to additional changes in physiological processes, such as the accumulation of starch and anthocyanins (Gebhard et al., 1990a).
Salvinia natans L. was effective in removing Hg(II) from wastewater. Senescence was seen at 5 ppm of Hg(II). Chlorophyll, Hill activity, protein, RNA, dry weight, and activities of catalase and protease decreased, whereas free amino acid content, peroxidase activity, and the ratio of acid to alkaline pyrophosphatase activity increased (Sen & Mondal, 1987). The effects of different forms of mercury on other plants are summarized in Table I.
Mercury, at a low concentration (3 [micro]M), enhanced the intensity of room-temperature fluorescence emitted by phycocyanin and induced a blue shift in the emission peak of Spirulina cells, indicating alterations in the energy transfer within the phycobilisomes. Selective bleaching of the b-84 chromophore of pbycocyanin was induced by mercury. The differential effect of mercury toward C-phycocyanin and allophycocyanin may be due to the difference in the protein conformation of the two compounds (Murthy & Mohanty, 1995). The interaction of divalent mercury with the light-harvesting proteins (LHC-II) of chloroplast thylakoid membranes was investigated in aqueous solutions with ion concentrations of 0.01-20 mM, using Fourier transform infrared (FTIR) difference spectroscopy. Strong mercury-ion binding was seen with different protein subunits at a very low metal-ion concentration, with drastic structural modifications of the interacting proteins. The major metal ion binding sites were those of the protein carbo nyl group, the nitrogen atom, or both, and the sulphur donor sites were also the target of mercury--protein complexation (Ahmed & Tajmir-Riahi, 1993).
The supply of reduced glutathione (GSH) and other thiols to excised greening leaf segments of Zea mays cv. Ganga 5 prevented the inhibitory effect of mercury on chlorophyll biosynthesis. The supply of GSH also increased the chlorophyll content, both in the absence and presence of mercury, after preincubation with either [HgCl.sup.2] or GSH. The concentration and time-dependent protective effect of GSH seem to be mediated by the restricted uptake of the metal-involving cytoplasmic protein synthesis (Jam & Puranik, 1993). With higher concentrations of mercury, glutathione transferase activity in seedlings of Zea mays cv. Tisa increased in the shoots and slightly decreased in their roots. The activity increased in roots three days after exposure to 30 [micro]M mercury (Komives et al., 1994).
Plants of Bacopa monnieri were treated with six different concentrations of mercury for 4, 7, and 14 days. The results suggest that an increase in cysteine, total -SH, reduced glutathione, and ascorbic acid content by mercury in the initial exposure period are part of the overall expression of mercury tolerance in the plant and that the decrease in chlorophyll protein content is a consequence of mercury toxicity at higher metal concentrations and increased exposure (Sinha et al., 1996). The membranes of plant and animal cells contain aquaporins, proteins that facilitate the transport of water. In plants, aquaporins are found in the vacuolar membrane (tonoplast) and the plasma membrane. Many aquaporins are mercury sensitive, and in AQP1 a mercury-sensitive cysteine residue (Cys-189) is present adjacent to a conserved Asn-Pro-Ala motif. A new Arabidopsis aquaporin, d-TIP (for tonoplast intrinsic protein) is located in the tonoplast. The water-channel activity of d-TIP is sensitive to mercury. Site-directed mut agenesis was used to identify the mercury-sensitive site in these two aquaporins as Cys-116 and Cys-118 for d-TIP and [gamma]-TIP, respectively. These mutations are at a conserved position in a presumed membrane-spanning domain not previously known to have a role in aquaporin mercury sensitivity. Comparing the tissue expression patterns of d-TIP with [gamma]-TIP and a-TIP showed that the TIPs are differentially expressed (Daniels et al., 1994, 1996).
Segments of the inner tissue (mostly ground-tissue parenchyma) from hypocotyls of Helianthus annus cv. Giganteum extended rapidly in water and could be reversed in a hypertonic osmoticum. The rates of water uptake or loss were sensitive to submillimolar concentrations of [HgCl.sub.2]. Turgor changes in the state of water channels were affected by [HgCl.sub.2], so that the mercury-sensitive sites are not accessible to mercaptoethanol (Hejnowicz & Sievers, 1996).
3. Genetic and Related Effects
For more than 60 years, numerous experiments have been carried out to study the genetic effects of mercury compounds in experimental test systems using a variety of genetic endpoints (see De Flora et al., 1994 for a review). In the earliest work on plant test systems, multinucleate cells were recorded in the root tips of corn seedlings exposed to solutions of New Improved Ceresan (containing ethyl mercury phosphate). Abnormal mitosis led to the formation of polyploid giant nuclei and micronuclei in these cells. C-mitosis and chromosome doubling were recorded after exposure to the fungicide Granosan (2% ethyl mercury chloride; Kostoff; 1939, 1940) of the germinating grains of Secale cereale, Triticum durum, T. persicum, T. polonicum, T. aegilopoides, and, to some extent, of Pisum sativum. No chromosomal alteration was observed in Linum usitatissimum or Crepis capillaris. However, inactivation of the spindle apparatus and polyploidy in Crepis capillaris and Avena sativa were recorded after exposure to phenyl m ercury dinaphthyl methane disulfonate. On exposure to solutions of phenyl mercuric hydroxide and phenyl mercuric nitrate, shoot apexes of the monocotyledons Zea mays, Anacharis canadensis, and Ruppia maritima and of the dicotyledons Colcus blumei and Raphanus sativus showed different types of somatic mutations (MacFarlane, 1956; MacFarlane & Messing, 1953), in addition to c-mitosis, inhibition of spindle formation, and other chromosomal alterations in Allium cepa roots (see De Flora et al., 1994).
The most noticeable and consistent effect of mercurials was the induction of c-mitosis through disturbance of the spindle activity, resulting in the formation of polyploid and aneuploid cells, and c-tumors. C-mitosis was induced at similar dosages of all organic mercurials tested, butyl mercury bromide being the most active (Fiskesjo, 1969). Organomercurials had been reported to be 200 times more potent than inorganic mercury (mercury bromide) in inducing c-mitosis (Fahmy, F. Y., 1951 of Ramel, 1969). C-mitosis was also induced in Vicia faba following exposure of roots to methyl mercury hydroxide (Ramel, 1972) and in Tradescantia (clone 02) and Vicia faba exposed to the fungicide Panogen 15 (2.31% methyl mercury dicyandiamide; Ahmed & Grant, 1972). On the other hand, BAL (2,3 dimercaptoethanol) inhibited c-mitosis (Ramel, 1969).
Micronuclei were increased in roots of Eichhornia crassipes following exposure to methyl mercury chloride (Panda et al., 1988) and in Allium cepa, to methyl mercury chloride (Panda et al., 1989). In Allium tests, [EC.sub.50] values for mercury were 9.0 x [10.sup.-7] M for MMC and 3.3 x [10.sup.-6] M for [HgCl.sub.2] (Fiskesjo, 1988). Salts, including mercuric chloride, when given to Allium cepa root tips, could, in varying degrees, cause different types of chromosome, nucleus, and nucleolus irregularities (Liu et al., 1995).
After exposure to inorganic salts of mercury in Allium cepa and Allium sativum, the mitotic index in the root-tip cells was reduced and the frequency of chromosomal aberrations was increased in degrees directly proportional to the concentrations used and to the duration of exposure to the mercurial. The period of recovery after removal of mercury was inversely related to the concentration and duration of exposure. The lowest effective concentration tested was 10 ppm. Cytotoxic effects of [HgCl.sub.2] were greater than were those of [Hg.sub.2][Cl.sub.2]. A. sativum was more resistant than was A. cepa, possibly due to the greater amount of heterochromatin in the former and to the presence of lower amounts of sulphur compounds with affinity for mercury in the latter. Hordeurn vulgare seeds were less effected than were A. cepa when exposed to [HgCl.sub.2] for a short period (M. Patra, pers. obs.).
Disorganization of microtubules did occur on exposure of cell-suspension cultures of Daucus carota to methyl mercury at higher concentrations (4-6 [micro]g/ml) in a concentration--time-dependent manner. Analyses of soluble protein and carbohydrate content, dry weight, and cell viability indicated inhibitory effects on cell metabolism. The observed disruption of plant-cell microtubules, induced by exposure to methyl mercury, may be secondary in response to an initial inhibition of synthetic pathways and membrane perturbations (Czuba et al., 1987).
B. LOWER PLANTS
The antimicrobial actions of mercury salts on bacteria may be a result of combination with essential sulphydryl groups. After treatment with mercuric chloride or phenyl mercury nitrate, the bacteria appear dead but are easily revived by active thiol-containing agents (Albert, 1973). Sulphur compounds without thiol groups do not show similar behavior (Sexton, 1963). Silver (1984) has published extensive information on the mechanism of microbial resistance to mercury. Organomercurials are more active as bactericides or fungicides than are the inorganic salts. The difference may partly be attributed to lipid solubility, where penetration of seed coats is required to reach a parasitic fungus (Sexton, 1963). The toxic action of mercurials may also be related to a nonspecific inhibition of a variety of intracellular enzymes and several specific thiol-containing respiratory enzymes in vitro.
Divalent inorganic mercury can also be reduced to elemental mercury directly by widely occurring Pseudomonas bacteria or yeasts. The alkyl mercury by microbial action in bottom sediments is biomagnified through the food chain (Beijer & Jernelov, 1978). Direct toxic effects of mercury were also observed on lower plants like Anacystis nidulans (Lee et al., 1992) and Pleurotus sajor-caju (Purkayastha et al., 1994).
Organic mercury compounds in bacterial test systems generally yielded negative or low results. The azo-dye mercury orange was not mutagenic in the reversion assay (spot test and plate test) with the Salmonella typhimurium his strains TA1535, TA1537, TA1538, TA98, and TA100, with or without S9 mix (Brown et al., 1978) and methyl mercury acetate in the plate test, with strains TA1535, TA1537, TA98, and TA100, with or without S9 mix (Bruce & Heddle, 1979). Methyl mercury chloride, in the absence of S9 mix, gave negative spot test results with the Salmonella strains TA1535, TA1537, TA1538, TA98, and TA100 and with the E. coli trp-strains WP2 and Wp2 WP2her (Kanematsu et al., 1980).
Triticum aestivum seedlings, raised from mercury-treated seeds, were spray inoculated with Cochliobolus sativus. All salts had strong inhibitory effects on spore germination and germ tube growth of the pathogen. Neither form of toxicity appeared to be linked with induction of resistance in wheat seedlings (Chakraborty & Sinha, 1989).
At low concentrations mercury has a toxic effect on the degrading capabilities of microorganisms. Two new Pseudomonas isolates capable of utilizing 2,4-D and 2,4,5-T as their sole C- and energy-source were isolated from a municipal sewage plant in the presence of mercury (Sebuktekin et al., 1987).
In the cultures of four yeasts--Saccharomyces cerevisiae, S. lipolytica, Candida tropicalls, and C. utilis--mercury was inhibitory in the concentration range 10[e.sup.-5] 10[e.sup.-4] M and increased the lag phase and generation time. Sensitivity to the metal was enhanced by a reduction in pH. All four strains showed a dose-related accumulation of mercury over a thousandfold range of concentrations in the medium (Hermenegild & Schwantes, 1983). Growth of Fusarium oxysporum f. sp. psidii was completely inhibited by 100 [micro]g/ml mercury in guavas, as was growth of Agrobacterium tumafaciens in Swiss apples (Dwivedi, 1991).
Rhizosphere fungi associated with Saccharum officinarum were effectively controlled by standard fungicides containing mercury, with the exception of Fusarium moniliforme [Gibberella fujikuroi] in black cotton soil (Peshney et al., 1994). Additional [Hg.sup.2+] inhibited the growth, oxygen evolution, and oxygen uptake (photosystem I, PSI) in the cells of Cylindroipermum and also the variable fluorescence of Chl a. Action sites of [Hg.sup.2+] are located prior to the electron-donation site of PMS, but possibly after the cytochrome reductase level (Singh et al., 1989).
Concentration- and time-dependent changes (predominantly decreases) were observed in the content of chlorophylls a and c, carotenoids, and pheophytins in marine phytoplankton exposed to mercury for 30 days in situ (Kulikova, 1987). In Chlorella vulgaris, mercury was highly toxic for nutrient ([NO.sub.3] and [NH.sub.4]) uptake. PS II was the primary site of action (Rai et al., 1991).
Extracellular a-galactosidase (E.C.18.104.22.168) from Aspergillus ficuum NRRL 3135 was inhibited by mercury (Zapater et al., 1990). Mercury also inhibited proteinase yscJ, a new yeast peptidase (Wagner & Wolf, 1992).
In Euglena gracilis, mercury decreased the activities of four enzymes involved in the fixation of [CO.sub.2] (carbon reduction cycle) for at least the first two days of exponential growth (De Filippis & Ziegler, 1993). Sublethal concentrations decreased levels of adenosine 5-triphosphate (ATP), especially in young cultures. Adenosine monophosphate shows only small changes. It is possibly the equilibrium between ADP and ATP that is affected by mercury via an inhibition of ATP-forming reactions (De Filippis et al., 1981).
Physiological responses of different phytoplanktons to mercury are similar (Gotsis-Skretas & Christaki, 1992; Hardstedt-Romeo & Gnassia-Barelli, 1988). In low-dose, long-term experiments (0.05-0.25 [micro]M [Hg.sup.2+], 10 days) with the filamentous [N.sub.2]-fixing cyanobacterium Nostoc calcicola Breb, photoautotrophic growth was severely inhibited, with concurrent loss of photosynthetic pigments (phycocyanin [greater than] chlorophyll a [greater than] carotenoids) and nucleic acids. The elevated [Hg.sup.2+] concentration irreversibly damaged the cell membrane. In high-dose, short-term experiments, the in vivo activities of selected enzymes (glutamine synthetase [greater than] nitrate reductase [greater than] nitrogenase) were less inhibited by [Hg.sup.2+] than was the uptake of nutrient ions (Singh & Singh, 1992). The tolerance to mercury by microorganisms was found to be in the order: total population [greater than] nitrogen fixers [greater than] nitrifiers (Semy et al., 1989).
Among prokaryotes, the [rec.sup.-] assay in Bacillus subtilis showed a slight difference in the inhibition zones produced by a compound identified as "HGCl" in strains H17 (wild type) and M45 ([rec.sup.-]; Kanematsu et al., 1980). Mercuric chloride induced a weak filamentation in the DNA repair-deficient E. coli strain AB2463 ([recA.sup.-]; Brandi et al., 1990). In the yeast Saccharomyces cerevisiae, inorganic mercury [unspecified Hg(II)] induced cytoplasmic respiration-deficient mutants under conditions of low cell viability in strain N123 and produced mitotic crossing over in convertants but not in revertants of strain D7 (Fukunaga et al., 1981).
In yeasts and molds the results are variable. Saccharomyces cerevisiae (strain not specified) methyl mercury induced "petite" [rho.sup.-] mutants in mitochondrial DNA and, weakly, mitotic nondisjunction. Methyl mercury chloride blocked the cell cycle in [G.sub.1] but induced mitotic crossing over in strain D7 (Phipps & Miller, 1982). It inhibited spontaneous [rho.sup.-] mutants but induced a significant number of erythromycin-resistant mutants (Phipps & Miller, 1983). Sucrose gradient analysis of DNA molecular weight in the slime mold Physarum polycephalum treated with dimethyl mercury showed a radiomimetic breakage that did not depend on DNA replication (Yatscoff& Cummins, 1975).
VI. Tolerance of and Resistance to Mercury
Tolerance is usually specific to one particular metal, although a single plant may possess one or more mechanisms that enable it to tolerate excesses of more than one element. Many species in several plant families have been found to show tolerance to heavy metals. In multi-cellular and compartmentalized organisms broad-spectrum mechanisms, which confer resistance to a number of different heavy metals, have evolved (for example, metallothioneins in animals and phytochelatins in plants and fungi). In the bacteria, resistance is often specific to one metal or a small number of related metals.
A. MICROBES AND LOWER GROUPS
Bacterial plasmids encode resistance systems for toxic metal ions, including [Hg.sup.2+]. Chromosomal determinants of toxic metal resistance are known. For mercury, the plasmid and chromosomal determinants are basically the same. The largest group of metal-resistance systems functions by energy-dependent efflux of toxic ions, through ATPases and chemiosmotic cation-proton antiporters (Silver, 1996; Silver & Phung, 1996). The molecular mechanism of bacterial resistance to organomercurials involves the novel enzyme organomercurylyase (Walsh, 1994).
Plasmid-determined resistances to mercury have been identified in high frequencies in natural populations of common bacteria. The inducible mercury resistance (mer) operon encodes both a mercuric ion uptake system and a detoxification enzyme. This system is also present in microorganisms with broad spectrum resistance to organomercury compounds, but they have, as well, another enzyme, which can split the covalent carbon-mercury bond in such compounds. Toxic-element resistance has potential applications in pollution abatement, metal leaching from ores, upgrading fossil fuels, biocatalysis, and agriculture (for example, as donors of genes to enhance plant resistance; Summers, 1985).
A set of mercury resistance plasmids was obtained from wheat rhizosphere soil via exogenous plasmid isolation by using Pseudomonas fluorescens R2f, Pseudomonas putida UWC1, and Enterobacter cloacae BE1 as recipient strains. The isolation frequencies were highest from soil amended with high levels of mercury. With P. putida UWC1 as the recipient, the isolation frequency was significantly enhanced in wheat rhizosphere. Twenty transconjugants, analyzed per recipient strain, contained plasmids 40--50 kb long. Eight selected plasmids were distributed among five groups, as shown by restriction digestion coupled with a similarity matrix analysis. However, all plasmids formed a tight group, as judged by hybridization with two whole-plasmid probes and comparisons with other plasmids in dot blot hybridization analyses. The results of replicon typing and broad-host-range incompatibility, including group-specific PCR suggested that the plasmid isolates were not related to any previously described Inc group. One plasmid, pEC10, transferred into a variety of bacteria belonging to the class Proteobacteria, mobilized as well as retromobilized the IncQ plasmid pSUP104. A PCR method for detection of pEC10-like replicons was used, in conjunction with other methods, to monitor pEC10-homologous sequences. The presence of mercury enhanced the prevalence of pEC10-like replicons in soil and rhizosphere bacterial populations (Smit et al., 1998).
The best-studied determinants of metal resistance are those of mercuric ion resistance in gram-negative bacteria. A periplasmic protein, MerP, an inner-membrane transport protein, MerT, and a cytoplasmic enzyme, mercuric reductase (the MerA protein), are responsible for the transport of mercuric ions into cell and their reduction to elemental mercury, Hg(II). The mercury-resistance proteins all contain motifs of paired cysteine residues, which are metal-binding regions. Direct binding of mercuric ions to the cystein pairs has not been demonstrated, but mutagenesis of cysteine residues in MerT and in mercuric reductase causes loss of resistance. The mercuric-reductase protein can be up to 6% of the soluble protein of the cell when mercury resistance is fully induced, and the protein is easily purified.
Typically, the genes for metal resistance occur in discrete determinants, and the proteins required for resistance are expressed from a metal-inducible transcriptional promoter on the DNA. Genetic fusions between these metal-inducible promoters and a "reported gene" have been proposed and patented as in vivo biosensors for the detection of heavy metals. Brown (1994) examined the induction of expression of the mercury resistance genes by coupling its transcriptional promoters (Pmer) to a suitable reporter gene (Rouch & Parkhill, 1994, unpublished data). The production of B-galactosidase by the lacZ gene expressed from Pmer was measured at different external mercuric-ion concentrations. Pmer has a steep "hypersensitive" induction curve, in which induction from 10% to 90% of full activity occurs across a fourfold range of concentration. Thus Pmer may only allow the threshold detection of mercuric ions, with little direct quantification possible. The hypersensitive response of Pmer may be typical of the response to purely toxic metals (mercury, cadmium, or arsenic).
Forty strains of Acinetobacter classified into four genospecies--A. baumannii (33 isolates), A. calcoaceticus (three isolates), A. junii (three isolates), and A. genospecies3 (one isolate)--were resistant to multiple metal ions. The maximum number of strains (60%) was found to be sensitive to mercury. A. genospecies3 was the most resistant species (Dhakephalkar & Chopade, 1994).
Mercury-resistant Rhizobium sp. in low numbers was isolated from plant nodules by selection on Mannitol Yeast Extract Agar media containing 8.4 [micro]g/ml [HgCl.sub.2]. Ten strains were obtained that were resistant to [HgCl.sub.2], of which five were also resistant to organomercurials. The rate of oxidation of NADPH was higher in the presence of [HgCl.sub.2], Hg(I) acetate, thiomersol, PMA, and p-hydroxy mercuribenzoate (Nath et al., 1993).
On being subjected to mutation using ethyl methyl sulphonate, 23 cultures of cowpea Rhizobium, very sensitive to mercury, gave a few mutant strains with a high degree of tolerance to both mercury and lead, which could produce root nodules on cowpea (Kesavan & Purushothaman, 1991).
Bacterial detoxification of mercury has been carried out in sediment microcosms (Rochelle & Olson, 1992). Cladosporium herbarum, isolated from Taif soil, was able to grow in liquid media containing up to 25 [micro]g/ml mercury, indicating its resistance to mercury (Hashem, 1993). Epsarium oxysporum was inhibited while screening natural materials on detoxification of mercury chloride (Kim et al., 1992). Microbial activities were also used to remove elemental mercury from mercury-contaminated wastewater from a nuclear weapon plant in Oak Ridge, Tennessee (Barkay & Turner, 1992). Thiobacillus ferrooxidans is an acidophilic chemoautotrophic bacterium sensitive to mercury ions that is used industrially in leaching metals from a variety of ores. A group of mercury-resistant T. ferrooxidans strains that volatilized mercury were isolated and characterized. The entire coding sequence of the mercury-on resistance gene was located in a 2.3 kb fragment of chromosomal DNA (encoding 56,000 and 16,000 molecular weight prot eins) from strain E--15 of Escherichia coil (Shiratori et al., 1989). A T. ferrooxidans E--15 mercury-reductase (EC--l.16.l.1) mer A gene, cloned E. coli DH5-a, contained 1,635 bp and shared a 78.2% and a 76.6% sequence homology with transposon Tn501 and plasmid R100 mer A genes, respectively. A 545 amino acid protein sequence was deduced, with an 80.6% and an 80.0% homology with transposon Tn501 and plasmid R100 gene products, respectively. Divergence among the three mer A sequences was clustered within a specific region at residues 41--87. By codon usage analysis, it was speculated that the T-ferrooxidans mer A gene originated from transposon 501, plasmid R100, or a common ancestral gene, but not from T. ferrooxidans itself. To express the T.ferrooxidans mercury resistance (merC) gene in Escherichia coli JM1O9, plasmid pTMC527 was constructed by cloning the mer operon promoter with intact merC and truncated merA genes into vector plasmid pKK223--3 under the control of the tac promoter. Transformants grew in Luria broth with 0.3% glucose, but not in plain Luria broth or in 0.1% glucose broth. Thus, expression of the merC gene affected host-cell growth. The MerC protein synthesized in E. coli had an N-terminal amino acid sequence, which agreed with that deduced from the nucleotide sequence, except that the initiating Met residue was absent. The MerC protein was localized in the particulate (membrane) cell fraction. E. coli cells carrying pTMC527 took up [[Hg.sup.2+].sub.203] in an IPTG-dependent manner (Inoue et al., 1989, 1996).
A broad-spectrum mercury-resistant Bacillus pasteurii strain, DR2, isolated from the effluents of Durgapur Steel Plant, India, grew well in nutrient broth containing PMA. [HgCl.sub.2] partially inhibited the growth. However, phenyl mercuric nitrate and other hydrophobic compounds facilitated the transport of glucose across the cell wall and thereby stimulated growth (Pahan et al., 1993).
Of 278 Salmonella strains resistant to mercury, four acrotypes--S. panama, S. typhimurium, S. agona, and S. oranienburg--were located from the effluents of two sewage-treatment plants in Rio de Janeiro, Brazil. Using standard Escherichia coli K 12 F-Nal-r strain and cultures isolated from sewage water (Citrobacter freundu Tc-r and Salmonella oranienburg Sm-r) as the recipients, Hg-r transconjugants were isolated at a rate of 86.4%. All trans-conjugants expressed the same level of resistance as the corresponding Salmonella (donor) strain (Nogueira et al., 1993). Tolerance of ethyl mercury chloride was found in populations of smut fungi on Triticum aestivum in Romania (Nedelcu et al., 1992). Resistance to mercury in cultures of 15 decomposer basidiomycete samples increased with the ability of the fungus to produce phenol oxidases, especially the intracellular tyrosinase (Hoiland, 1995).
B. HIGHER PLANTS
Certain higher plants have an armory of metal-protection mechanisms, including metal sequestration by specially produced organic compounds, sequestration in certain cell compartments, metal ion efflux, and organic ligand exudation. A set of heavy metal-complexing peptides was isolated from plants and plant-suspension cultures, with the structure [([gamma]-glutamic acid-cysteine).sub.n]-glycine (n = 2-1l)[[([gamma]-Glu-Cys).sub.n]-Gly]. These peptides appear upon exposure of all autotrophic plants and select fungi to metals of the transition and main groups of the Periodic Table and are called phytochelatins (PCs). The biosynthesis of PCs proceeds by metal activation of a constitutive enzyme, [gamma]-glutamylcystenine dipeptidyl transpeptidase, given the trivial name PC synthase, and catalyzes the reaction [gamma]-Glu-Cys-Gly + ([gamma]-Glu-Cys-Gly) leading to [([gamma]-Glu-Cys).sub.n+1]-Gly + Gly. The substrate used is glutathione. The plant vacuole is the transient storage compartment for these peptides, which probably dissociate, and the free peptide is subsequently degraded. Sequestration of heavy metals by PCs confers protection for heavy metal-sensitive enzymes. Higher plants and Schizosaccharomyces pombe respond to heavy-metal stress of cadmium and mercury by synthesizing PCs that act as chelators. The yeast hmt I gene, encoding a membrane transport protein, is essential for its PC-mediated heavy-metal tolerance (Ortiz, 1994).
Optical spectroscopy and reverse-phase HPLC showed that pH did not influence the Hg(II)-binding capacity of these peptides. The reverse-phase HPLC assays indicated a rapid transfer of Hg(II) from glutathione to PCs and from shorter- to longer-chain PCs. The strength of Hg(II) binding to glutathione and phytochelatins followed the order: [gamma]-Glu-Cys-Gly[less than][([gamma]Glu-Cys).sub.2]Gly[less than][([gamma]-Glu-Cys).sub.3]Gly[less than][([gamma]-Glu-Cys).sub.4]Gly (Mehra et al., 1996). Glutathione and phytochelatin played definite roles in resistance in Hydrilla verticillata (l.f.) Royle & Vallisneria spiralis L. under mercury stress (Gupta et al., 1998).
In Rauwolfia serpentina suspension cells, heavy-metal ions entering cells at sublethal concentrations are totally complexed by PCs (and to a much lesser extent to some high-molecular-weight proteins). A series of metal-sensitive plant enzymes--such as alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, nitrate reductase, ribulose-l ,5-diphosphate carboxylase, and urease--tolerate [Cd.sup.2+] and also [Hg.sup.2+] in the form of a PC complex from 10 to 100 times greater than the free-metal ion. Free PC peptides reactivate metal-poisoned nitrate reductase in vitro up to a thousand times better than do chelators such as glutathione and citrate (Kneer & Zenk, 1992). In root cultures of Rubia tinctorum, all metal species induced PCs to various degrees and also their desglyl peptides (Maitani et al., 1996).
Suspension cultures of haploid tobacco (Nicotiana tabacum) cells were subjected to muta-genesis using 0.3-0.6% ethyl methane sulfonate. When resistant green plantlets were transferred to a rooting medium with [HgCl.sub.2], only three plantlets in the 0.025 mM series survived, but they developed no roots (Koo, 1982). Both resistance and demethylation to methyl mercury were modified by light and/or 2,4-dichlorophenoxyacetic acid (Czuba, 1987). [HgCl.sub.2]-tolerant variants were elected from nitrosoguanidine (NTG)-treated suspension cell cultures of cow pea (Vigna unguiculata and V sinensis) initiated from hypocotyl callus and incubated with 18 [micro]g/ml [HgCl.sub.2]. Most [HgCl.sub.2]-tolerant calli were recovered from cells treated with 10 [micro]g/ml NTG. Mercury tolerance was not lost during long-term culture in the absence of mercury stress, but the number of tolerant calli dropped from 89 to 9 after 140 days (Ahmed et al., 1993).
Experiments have been carried out to develop mercury-tolerant plants through previous exposure to low doses of mercury and subsequent planting of the next generation in mercury-contaminated soil. In four pasture plants of Uttar Pradesh (India), during germination stage, legumes (Indigofera enneaphylla and Desmodium triflorum) were most tolerant (Maury et al., 1986).
Seeds of Hordeum vulgare were exposed to concentrations of ethyl methane sulfonate (EMS), maleic hydrazide (MH), methyl mercuric chloride (MMC), and mercury-contaminated soil. Residual mercury in seeds conferred protection against the genotoxicity of these agents. Preexposing the mercury-treated seeds to buthionine sulfoximine, an inhibitor of PC synthesis, significantly prevented the genotoxic adaptation to MH and MMC. Compared with normal seedlings, the seedlings grown from treated seeds exhibited a higher amount of non-protein SH. These findings indicate a possible involvement of phytochelatins in the mercury-induced adaptive response (Subhadra et al., 1993). Pretreatment of barley seeds with 10 ppm [HgCl.sub.2] was followed by sowing. The harvested seeds were subjected to 100 ppm [HgCl.sub.2] before sowing in the next generation. Both cytotoxic and agronomic alterations were reduced, indicating that pretreatment with low doses of [HgCl.sub.2] confers a certain degree of resistance to further exposure wit h higher levels of [HgCl.sub.2] (M. Patra, pers. obs.).
Chloris barbata and Cyperus rotundus from a mercury-contaminated site near a chloralkali plant at Ganjam, India, exhibited high tolerance to mercury compared with the same species from a noncontaminated site. Tolerance to mercury was higher in Chloris barbata than in Cyperus rotundus (Lenka et al., 1993).
Phytoremediation involves the use of plants to extract, detoxify, and/or sequester environmental pollutants from soil and water. The heavy-metal burden with which tree roots have to cope originates either from natural metalliferous soils or from continual manmade inputs into forest ecosystems. Heavy-metal toxicity also includes assessment of the disturbance of the mineral nutrition of tree roots. Counteracting strategies include phytochelatin response, tolerance mechanisms, and raising of the rhizosphere pH in relation to tree species and mycorrhizas (Kahle, 1993).
Plants that can process heavy metals may provide efficient and ecologically sound approaches to sequestration and removal. MerA converts toxic [Hg.sup.2+] to less toxic, relatively inert [Hg.sup.0]. The bacterial merA sequence is rich in CpG dinucleotides and has a highly skewed codon usage, both of which are particularly unfavorable to efficient expression in plants. A mutagenized merA sequence, MerApe9, modifying the flanking region and 9% of the coding region, was constructed and placed under the control of plant-regulatory elements. Transgenic Arabidopsis thaliana seeds expressing merApe9 germinated. These seedlings grew, flowered, and set seed on a medium containing [HgCl.sub.2] concentrations of 25-100 [micro]M (5-20 ppm), levels toxic to several controls. Transgenic merApe9 seedlings evolved considerable amounts of [Hg.sup.0] relative to control plants. The rate of mercury evolution and the level of resistance were proportional to the steady-state mRNA level, confirming that resistance was due to expr ession of the MerApe9 enzyme. These and other data suggest that there are potentially viable molecular genetic approaches to the phytoremediation of metal-ion pollution (Rugh et al., 1996a, 1996b, 1996c; Wilde et al., 1994). A problem that is more serious than ionic mercury is the methyl mercury produced by native bacteria at polluted sites, which is concentrated up the food chain and poses the most immediate threat to wildlife and human populations (Meagher & Rugh, 1997). When tested with a mercury-vapor analyzer, transgenic plants grown on a [HgCl.sub.2] medium released volatile mercury in the air, suggesting that these plants were converting the toxic mercury supplied in the nutrient medium to a vapor form. Such reduction to nonionic form of mercury was seven times more in transgenic plants than in the control plants.
In order to explore the potential of plants to extract and detoxify methyl mercury, Arabidopsis thaliana was engineered to express a modified bacterial gene, merBpe, encoding MerB under control of a plant promoter. MerB catalyzes the protonolysis of the carbon--mercury bond, removing the organic ligand and releasing Hg(II), a less mobile mercury species. Transgenic plants expressing merBpe grew vigorously on awide range of concentrations of organic mercurials, where plants lacking the gene were severely inhibited or died. Six independently isolated transgenic lines produced merBpe mRNA and MerB protein at levels that varied over a 10-15-fold range. Even the lowest levels of merBpe expression conferred resistance to organomercurials. The work suggests that native macrophytes, such as trees, shrubs, and grasses, that are engineered to express merBpe may be used to degrade methyl mercury at polluted sites and to sequester Hg(II) for later removal. Transgenic Arabidopsis plants expressing the bacterial merB gene are resistant to methyl mercury, whereas control plants die. Transgenic seeds germinate, grow, and flower at nearly the rate of unchallenged controls, when grown on toxic levels of organic mercury compounds (Bizily et al., 1999).
The ability of yellow poplar (Liriodendron tulipifera) tissue cultures and plantlets to express modified mercuric reductase (merA) gene constructs was examined. Mercury-resistant bacteria express MerA to convert highly toxic, ionic mercury, Hg(II), to elemental mercury, Hg(0), Because alteration of the bacterial merA gene sequence was necessary for high-level expression in Arabidopsis thaliana, yellow poplar proembryogenic masses (PEMs) were transformed with three modified merA constructs via microprojectile bombardment. Each construct was synthesized to have altered flanking regions with stepwise increases (0%, 9%, and 18% blocks) of modified coding sequence. All merA constructs conferred resistance to toxic, ionic mercury for independently transformed PEM colonies. The stability of merA transgene expression increased in parallel with the extent of gene-coding sequence modification. Regenerated plantlets containing the most modified merA gene (merA 18) germinated and grew vigorously in media containing norm ally toxic levels of ionic mercury. The merA 18 plantlets released elemental mercury at approximately ten times the rate of untransformed control plantlets. These results indicate that plants expressing modified merA constructs may provide a means for the phytoremediation of mercury pollution (Rugh et al., 1998).
Transgenic plants cleave mercury ions from methyl mercury complexes, reduce mercury ions to the metallic form, take up metallic mercury through their roots, and evolve less toxic elemental mercury at concentrations well below OSHA standards. The result is a costeffective, permanent, aesthetically pleasing, and in situ solution, which meets or exceeds regulatory standards while reducing mercury toxicity by a factor of 10,000.
Genetically engineered plants contain modified forms of bacterial genes that break down methyl mercury and reduce mercury ions. The first gene successfully inserted into plants was merA, which codes for a mercuric-ion reductase enzyme, reducing ionic mercury to the less toxic elemental form. MerR codes for an organomercurial lyase protein that cleaves mercury ions from highly toxic methyl mercury compounds. Plants with the merB gene have been shown to detoxify methyl mercury in soil and water. Both genes have been successfully expressed in Arabidopsis thaliana, Brassica (mustard), Nicotiana tabacum (tobacco), and Liriodendron tulipifera (tulip poplar). Plants currently being transformed include cattails, wild rice, and Spartina, another wetland plant (Phytoworks, 1997).
Other methods of phytoremediation include removal of mercury by accumulator plants, such as Typha sp. from wastewater (Krishnan et al., 1988) and Eichhornia crassipes. The chlorophyll content of E. crassipes gradually decreases with increasing concentrations of mercury (James et al., 1992). This species was employed to assess the bioconcentrate and genotoxicity of aquatic mercury in water contaminated with mercuric chloride or PMA at 0.001-1.0 mg/I (Lenka et al., 1990). Hg(II) was also removed from aqueous substrates by Ix-ora coccinea. Appreciable metal-ion sorption occurred at 2,000 ppm at pH 4.0-6.5 for Hg(II) (Tikku et al., 1990).
The problem of mercury contamination can be reduced appreciably by combining the standard methods of phytoremediation--removal of mercury from polluted areas through scavenger plants--with raising such plants both by routine mutagenesis and by genetic engineering. The different transgenics raised utilizing the two genes merA and merB are very hopeful prospects.
The authors are grateful to Prof. A. K. Sharma, Centre of Advanced Study, Department of Botany, University of Calcutta, and Dr. M. K. Majumder, University of Calcutta, for suggestions and advice. They would also like to thank the Ministry of Forest and Environment, Government of India, for partial financial assistance.
VIII. Literature Cited
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