The myriad of parameters regulating the chemical fate of specific elements in soils determine their solubility and availability for plant uptake. The plant uptake of chemical species in soil solution is also dependent on a number of plant factors. These include: physical processes such as root intrusion, water, and ion fluxes and their relationship to the kinetics of metal solubilization in soils; biological parameters, including kinetics of membrane transport, ion interactions, and metabolic fate of absorbed ions; and the ability of plants to adapt metabolically to changing metal stresses in the environment.
Physical Aspects of Ion Replenishment in the Rhizosphere
The relative efficiency with which plants harvest both essential nutrients and nonnutrients from soil is dependent in part on the interrelationships between plant and soil physical factors. The process of plant root intrusion within the soil profile provides an extensive rhizosphere for ion absorption. Dittmer (25) has shown that after 4 months of growth in a 0.052 m3 container of soil, the roots of winter rye had a surface area of 639 m2 and a combined length of 623 km. Although this provides an effective absorptive surface in contact with soil particles and associated soil solution, the concentration of individual ions in solution can be rapidly depleted by plant uptake. Depletion of ions in the rhizosphere is alleviated to some extent by diffusion of ions, and by mass flow of both water and ions from surrounding soil induced by transpirational demand of the plant (26). Ultimately, the supply of ions within the rhizosphere is controlled by the kinetics of solubilization of ions sorbed to the solid phase of soil, as discussed above, and the kinetics of removal by the plant root. Barber and Claassen (27, 28) have developed mathematical models to describe metal uptake by plants based on the above kinetic parameters. In effect, the elemental composition of the plant reflects to some extent, the composition of the soil solution; this represents a key factor in our understanding of the uptake behavior of metals.
Kinetic Parameters Regulating Plant Absorption of Metals
Over the past several years the authors and coinvestigators have studied the behavior of 15 trace metals in plants. These studies have indicated that: abiotic and biotic soil processes controlled the solubility and availability of metals for plant uptake; metals were taken up by plants at differing rates; and metals, once absorbed, varied as to their mobility within the plant, suggesting a second point of metabolic regulation. The complexities involved in attempting to employ plants as indicators of environmental pollution are illustrated by the results of investigations to compare the bioavailability of a number of endogenous soil elements and soluble amended metals (Table 2). It should be noted that the reported concentration ratios (CR values) are based on the total endogenous soil concentration of each element and on total metal amended (2.5 ppm). Only a small fraction of the endogenous metal is soluble and therefore available; similarly, although amended metals were supplied in soluble forms, solubility of nonvolatiles ranged from
A comparison of CR values for amended metals (Table 2), indicates a broad range in plant availability, especially considering the proportion of amended metal which is soluble. Relatively higher CR values are not only obtained for nutrient species (Mo, Mn, Cu, and Zn) but also for nonnutrient species such as Pb, Ni, Cd, Tl, As, and Sn. This suggests that the uptake of nonnutrient elements may be metabolically facilitated.
Essential nutrients exhibit two types of distribution in shoot tissues: relatively uniform distribution with leaves being the major site of deposition and transport within the plant through passive movement in the xylem and initial uniform shoot distribution, with remobilization of specific elements from leaves through phloem transport during senescence, to either developing leaves and/or seeds. The distribution of Cd, Hg, Sn, and Tl (Table 2) is similar to nutrient species such as Mg, K, Cu, and Mo, reflecting the potential for remobilization from senescing tissues. Nickel is readily remobilized from senescing tissues and accumulated in seeds; a tendency which is shared by a number of nutrilites, i.e., Fe, Cu, Mn, and Zn. Although not as obvious as Ni, Cd also exhibits a tendency for accumulation in seeds of soybean. The elements Ag and Cr are not very mobile within the plant and accumulate in lower stems. Since distributions of specific ions may vary, tissue selection becomes important when employing plants as monitor systems.
Complexation of ions may be the physiological mechanism responsible for the mobility of ions in the plant (11). In the case of nutrient species, Ca, Fe, Cu, Mn, and Zn can be shown to exist within the plant as organometallic complexes. Complexation may provide a basis for maintaining the solubility and mobility of chemically reactive species, permit conservation of substrates by allowing for remobilization, and provide a means of compartmentalization. In addition, complexation of nonnutrients may represent a mechanism for detoxification. This aspect will be addressed in conjunction with accumulator or indicator species. Although it is generally conceded that the uptake of nutrilites by plants is metabolically regulated, there is some question as to the mechanisms controlling the absorption of nonnutrient species. Much of the information required to understand the behavior of metal pollutants in plants can be extrapolated from an extensive data base available for nutrient species. Peculiar to higher plants is a pattern of ion uptake referred to as multiphasic uptake. Basically, as the concentration of an ion surrounding the root is increased, uptake or absorption by the plant exhibits a series of distinct isotherms, each of which has different kinetic characteristics. These kinetic constants, Km and Vmax, describe the affinity of the transport mechanism for a given ion and also the rate of uptake at half-saturating ion concentrations. Table 3 lists kinetic constants for the transport of a number of nutrient species. Based on these data, several points can be made concerning the uptake potential of plants. First, each ion exhibits a number of distinct kinetic phases; which based on corresponding Km values indicates that the plant root possesses the affinity to absorb ions over a broad concentration range. For the data shown this represents a concentration range of three to four orders of magnitude. Secondly, a comparison ofKm values and potential uptake rates (Vmax) indicate that there is a degree of control exerted in ion uptake which is related to the concentration of ions in soil solution. In effect, uptake is much more efficient at lower soil concentrations than at higher concentrations, for example, in the case of Mg (phases 1 and 2) a five fold increase in substrate ions results in less than a doubling of uptake rate. Finally, the consistency of this kinetic relationship for all nutrient ions studied, suggests that nonnutrient metals may behave similarly. Although uptake mechanisms can be shown to be quite specific for individual ions, competition with respect to absorption can be shown for groups of closely related anions and cations. Vange et al. (35) have shown that sulfite, thiosulfate, molybdate, selenate, and chromate competitively inhibit sulfate uptake and therefore behave as analogs of sulfate, while divalent phosphate, perchlorate and periodate do not affect sulfate transport. Similar relationships have been shown for potassium-rubidium (36) and copper-zinc (37, 38). Since higher plants are known to accumulate nonessential elements in appreciable quantities (39), these elements may be accumulated by mechanisms in place for absorption of chemically similar nutrilites. Unfortunately, little effort has been expended to understand the mechanisms regulating metal uptake by plants.
Since it can be readily shown that nonnutrient elements are accumulated by plants, what are the controlling mechanisms? Cutler and Rains (40) investigated the mechanisms of Cd uptake in excised roots of barley and concluded that absorption was nonmetabolic with uptake being the result of diffusion coupled to sequestration. However, these studies have several shortcomings. The Cd concentrations employed ranged from 1 to 20 ppm in solution, which is two to three orders of magnitude higher than would be encountered in soil solution, and well beyond physiological concentrations, especially if Cd were behaving as an analog of a required trace element. At high solution concentration of Cd, sorption may far outweigh the fraction of Cd being absorbed into the symplast. Finally, high Cd levels, especially with excised roots, may further have an inhibiting effect on metabolism and therefore permeability.
Although the work of Cutler and Rains (40) indicated that Cd uptake by plants was not metabolically regulated, the work of Vange et al. (35) suggests, as discussed earlier, that there are specific interactions in the absorption of nutrient and nonnutrient anions. Studies of the absorption of nonnutrient elements by the authors and coworkers have concentrated on Cd, TI, and Ni. Since the absorption of micronutrients by plants saturates carrier systems at -200 uM, the kinetic studies were performed below this concentration. Many of the problems associated with Cd absorption (40) were resolved when Cd concentrations were limited to
These exhibited two- and three-phase transitions, respectively. Replots of the individual isotherms shows each to conform to Michaelis-Menten kinetics as in the case of Cd (Table 4). These kinetic constants, especially in the case of Ni, indicate that the plant root possesses effective and efficient mechanisms for accumulating nonnutrient ions over a broad range of metal concentration in soil solution as evident for nutrient ions (Table 3) and suggests that similar mechanisms may be in place for uptake of other heavy metals. The saturation behavior of ion absorption processes is important to the use of plants as monitor systems since the concentration of a pollutant in soil solution is not directly related to plant content. In addition, the multiphasic behavior results in nonlinearity of response between soil solution concentration of an ion and plant absorption. Since the absorption of Cd, Tl, and Ni appears to be under metabolic regulation, ion competition studies were employed to determine if the absorption of these nonnutrilites resulted from behavior as analogs of essential nutrients. The interactions of a number of nutrient ions with Cd, TI and Ni have been determined (Table 5). The absorption of Cd from 0.1 ttM solutions was reduced by 17-33% in the presence of 0.5 ,LM Fe, Mn, Zn, and Cu. Detailed kinetic analyses of the mechanism of inhibition failed to resolve the nature of the interactions between Cd, Fe, and Mn but Zn and Cu do appear to be competitive inhibitors of Cd transport. Experimental variability due to physical adsorption of Cd prevented calculation of inhibitor constants (Ki). The TI absorption was inhibited by 57% in the presence of a 10-fold higher concentrations of K. Detailed kinetic analysis of the interactions using concentrations within phase 1 (Table 5) demonstrated that K was a noncompetitive inhibitor of TI transport. The calculated Ki value of 23 ,uM for classical noncompetitive inhibition indicates that a concentration of 23 uM K will reduce TI absorption by 50o at all concentrations within the concentrations bracketed by phase 1. Although not determined, a similar situation can be expected to occur for phase 2 at its Ki value. The absorption of Ni was inhibited by 25-42% in the presence of Cu, Co, Fe, and Zn. Detailed kinetic analyses failed to resolve the interaction of Co and Fe with Ni; however, both Cu and Zn were shown to be competitive inhibitors of Ni transport. The Km and Ki constants for Ni in the absence and presence of Cu were calculated to be 6.1 and 0.2 ,uM, respectively, while Km and Ki constants for Ni in the absence and presence of Zn were 6.7 and 24.4 ,uM, respectively. Since the Ki values for Cu and Zn represent the inhibitor concentration necessary to double the slope of the 1/v versus 1/[S] plot, Cu is shown to be a better competitor of Ni and Zn. The affinity of these carrier sites for Cu is about 60%'o of that for Ni, while their affinity for Zn is only 25% that of Ni. The kinetic parameters indicate that at a constant soil solution concentration of Cu and/or Zn, Ni uptake by plants will decrease as the Ni concentration is reduced through the concentration range comprising phase 2 (Fig. IC).
A kinetic approach to the behavior of heavy metal in soils and plants, aside from providing an insight into controlling mechanisms, uptake potential, and ion associations, is useful in interpreting and identifying some of the current problems associated with environmental dissemination of heavy metals from anthropogenic activities. For example, it is clear that the use of municipal sludge amendments for agricultural soils will result in higher plant concentrations of several heavy metals, but, in addition, amendments may also reduce the uptake of required nutrient analogs; similarly, the concentration of nutrient analogs in soil solution will affect the rate of absorption on nonnutrients.
Metabolic Behavior of Metals in Plants as Related to Tolerance
The feasibility of employing higher plants as monitors of metal pollution is dependent upon an understanding of the metabolic processes which enable plants to acquire needed nutrients and tolerate increasing levels of toxic elements. Plant breeding studies have led to the realization that many aspects of mineral nutrition are under genetic regulation and therefore governed by selection. Genetic control has been shown to govern the initial absorption of ions (41, 42) the oxidation-reduction of Fe (43), compartmentalization of ions within the root (44), transfer from root to xylem (45), and metabolic utilization (46, 47). Similarly, tolerance of plants to high levels of both nutrient and nonnutrient elements appears to be genetically controlled (48, 49). Tolerance of plants to individual metals, although forming the functional basis for the behavior of "indicator" or "accumulator" plants, is often misconceived. In the case of accumulator species, their adaptation of geographical areas containing high concentrations of endogenous metals involved genotypic evolution and selection over a period of time induced by the specific habitat. Man-made alterations in the environment, such as those resulting from mining operations, involve similar selection pressures, as is evident from cursory examination of these sites. Although the number of ecotypes is limited, the process of adaptation is relatively rapid. This would suggest that the indigenous wild population contains sufficient genetic variability to produce individuals capable of withstanding these adverse conditions.
Apparent plant tolerance can result from exclusion of toxic elements or metabolic tolerance to specific elements; the latter appears to be the more prevalent mechanism. Mechanisms for exclusion have been shown to include low root cation exchange capacities limiting uptake of Al and Mn (49), sorption of Zn to cell walls (50), and precipitation of Al by hydroxyl ions at the root surface (51, 52). Metabolic adaptation as a mechanism of metal tolerance in plants appears to be the rule rather than the exception. A comparison of individual grass species (Agrostis tenuis) collected from old mine dumps and adjacent pasture lands and stressed under laboratory conditions, resulted in identification of important traits of tolerant species (53). Plant populations derived from environments containing elevated levels of lead were tolerant to lead but not other metals; similar results were obtained for plants tolerant to other metals, suggesting that tolerance is specific. The observed tolerance of adapted plants and the sensitivities of plants growing in adjacent areas of low metal burden were not lost on cultivation although individuals did differ, and seeds collected from the tolerant population had the same tolerances as the parent and vegetatively propagated plants.
What metabolic changes in tolerant species could account for reduced metal toxicity? The major mechanisms appear to be compartmentalization, complexation and metabolic adaptation. Compartmentalization would provide a means of limiting the presence of toxic metals at cellular locations where toxicity is initiated. For example, Al effects are most pronounced on processes of cell division and respiration; and compartmentalization or exclusion from these metabolic sites, provides, an effective protective mechanism (54). A plant adaptation which may play a role to tolerance is the alteration of metabolic sequences to allow an organism to function in an apparently normal manner in the presence of large amounts of heavy metals (55). To date, modification in metabolic pathways has been demonstrated in Cu tolerant yeast and bacteria, but this mechanism has not yet been reported to occur in higher plants.
Complexation may also serve to alleviate the toxicity of heavy metals in plants. It appears to be a common process in plants, and therefore may also represent a common mechanism for tolerance to toxic metals. Investigations of the forms of nutrient cations such as Fe, Cu, and Zn in leaf tissues of agronomic species (56, 57) have shown these to be pr,esent as anionic complexes. Similarly, studies of the chemical forms of trace elements transported within the xylem (11, 25, 37-39, 58-62) and phloem (63) show Ca, Fe, Ni, Mn, and Zn to be transported as organic complexes. In the case of nutrient ions, complexation may provide a basis for maintaining their mobility within the plant and allowing for their accumulation at sites of metabolic use. A number of studies have dealt with the chemical form of Ni in leaves of accumulator species (64-66) and agricultural plants (67, 68). These have shown Ni to exist in low molecular weight cationic and anionic complexes, and therefore, may represent an example ofthe role of complexation in tolerance.
Plants as Monitors of Metal Pollution
In order for plants to serve as indicators of incremental increases of metals in soil, deposition must be sufficient to significantly increase the soluble, and ultimately, the plant-available, fraction in soil. Furthermore, increases must be distinguishable from changes in the soluble fraction which occur with time and weathering of minerals. These requirements are complicated by the broad range in total metal concentrations and form in soil and the environmental conditions under which the soils exist.
Studies to date indicate that increased soil levels resulting from diffuse sources such as stack emissions from coal combustion may not be detectable by measuring plant uptake and effects. Increases in soil-metal levels may be significant in cases of localized pollution such as in disposal of bottom ash and scrubber wastes from fossil fuel combustion, industrial wastes and municipal sewage sludges resulting in detectable increases in plants. However, even in situations of significant increases in metal levels, the wastes may, in themselves, modify soil properties influencing metal solubility and the value of plants as monitors of pollutant levels will be dependent on a detailed understanding and precise measurements of influential soil and plant variables. In the case of soil there is a need to understand those factors influencing solubility and form of the metals with times. In the case of plants, information is required regarding physical processes, membrane transport (ion interactions, kinetics) and metabolic fate, particularly as influenced by metal stress. Membrane transport will result in increased levels of metals within the plant based on available concentrations in soil solution. This can lead to a lO-lOOOx concentration by plants, at least for nutrient species, improving the feasibility for the use of the plant for detection of changes in environmental levels. On the basis of Cd, TI, and Ni behavior, it would appear that metals may exhibit similar tendencies. However, as demonstrated by ion competition studies, the uptake of these nonnutrient species will not necessarily be proportional to soil solution concentration, particularly at environmental concentration levels, and will be attenuated by the presence and concentration of their respective nutrient analogs. Once absorbed, the metabolic fate of metals will determine, not only their partitioning between root and shoot, but their toxicity. Although partitioning will undoubtedly vary with the element, toxicity should not be a major consideration at most environmental concentrations. In summary, for specific sites with local pollution sources, plants may be useful monitors of increases in metals, but this will require a specific understanding of soil solubilization and plant uptake mechanisms.
This word was performed under contract 21 1B00844 with the National Institute of Environmental Health Sciences.