Plant Physiol. (1998) 118: 219-226

Characterization of Zinc Uptake, Binding, and Translocation in Intact Seedlings of Bread and Durum Wheat Cultivars

Jonathan J. Hart^*, Wendell A. Norvell, Ross M. Welch, Lori A. Sullivan¹, and Leon V. Kochian

Plant, Soil, and Nutrition Laboratory, United States Department of Agriculture-Agricultural Research Service, Cornell University, Ithaca, New York 14853

	ABSTRACT

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Durum wheat (Triticum turgidum L. var durum) cultivars exhibit lower Zn efficiency than comparable bread wheat (Triticum aestivum L.) cultivars. To understand the physiological mechanism(s) that confers Zn efficiency, this study used ⁶⁵Zn to investigate ionic Zn²⁺ root uptake, binding, and translocation to shoots in seedlings of bread and durum wheat cultivars. Time-dependent Zn²⁺ accumulation during 90 min was greater in roots of the bread wheat cultivar. Zn²⁺ cell wall binding was not different in the two cultivars. In each cultivar, concentration-dependent Zn²⁺ influx was characterized by a smooth, saturating curve, suggesting a carrier-mediated uptake system. At very low solution Zn²⁺ activities, Zn²⁺ uptake rates were higher in the bread wheat cultivar. As a result, the Michaelis constant for Zn²⁺ uptake was lower in the bread wheat cultivar (2.3 µM) than in the durum wheat cultivar (3.9 µM). Low temperature decreased the rate of Zn²⁺ influx, suggesting that metabolism plays a role in Zn²⁺ uptake. Ca inhibited Zn²⁺ uptake equally in both cultivars. Translocation of Zn to shoots was greater in the bread wheat cultivar, reflecting the higher root uptake rates. The study suggests that lower root Zn²⁺ uptake rates may contribute to reduced Zn efficiency in durum wheat varieties under Zn-limiting conditions.

	INTRODUCTION

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Soils that contain insufficient levels of the essential plant micronutrient Zn are common throughout the world. As a result, Zn deficiency is a widespread problem in crop plants, especially cereals (Graham et al., 1992). The importance of plant foods as sources of Zn, particularly in the marginal diets of developing countries, is well established (Welch, 1993). The development of crop plants that are efficient Zn accumulators is therefore a potentially important endeavor. In addition to its effects on nutrition, Zn deficiency in crops is relevant to other areas of human health. Another consequence of Zn-deficient soils is the tendency for plants grown in such soils to accumulate heavy metals. For example, in the Great Plains region of North America, where soil Zn levels are low and naturally occurring Cd is present, durum wheat (Triticum turgidum L. var durum) grains accumulate Cd to relatively high concentrations (Wolnik et al., 1983). The presence of Cd in food represents a potential human health hazard and, in response, international trade standards have been proposed to limit the levels of Cd in exported grain (Codex Alimentarius Commission, 1993). Thus, there is a need to understand the physiological processes that control acquisition of Zn from soil solution by roots and mobilization of Zn within plants.

It has been demonstrated in recent years that crop plants vary in their ability to take up Zn, particularly when its availability to roots is limited. Zn efficiency, defined as the ability of a plant to grow and yield well in Zn-deficient soils, varies among wheat cultivars (Graham and Rengel, 1993). In field trials, durum wheat cultivars have been shown to be consistently less Zn efficient than bread wheat (Triticum aestivum L.) cultivars (Graham et al., 1992). Similarly, durum wheat varieties were reported to be less Zn efficient than bread wheat varieties when grown in chelate-buffered hydroponic nutrient culture (Rengel and Graham, 1995a).

The physiological mechanism(s) that confers Zn efficiency has not been identified. Processes that could influence the ability of a plant to tolerate limited amounts of available Zn include higher root uptake, more efficient utilization of Zn, and enhanced Zn translocation within the plant. Cakmak et al. (1994) showed that a Zn-inefficient durum wheat cultivar exhibited Zn-deficiency symptoms earlier and more intensely than a Zn-efficient bread wheat cultivar even though the Zn tissue concentrations were similar in both lines, suggesting differential utilization of Zn in the two cultivars. Rates of Zn translocation to shoots were shown to vary among sorghum cultivars, although correlations with Zn efficiency were not established (Ramani and Kannan, 1985). Root uptake kinetics have been reported to vary between rice cultivars having different Zn requirements, with high-Zn-requiring cultivars exhibiting consistently higher root uptake rates (Bowen, 1986). In contrast, a correlation between Zn efficiency and rates of root Zn uptake in bread and durum wheat cultivars could not be demonstrated (Rengel and Graham, 1995b).

In grasses Zn influx into the root symplasm has been hypothesized to occur as the free Zn²⁺ ion (Halvorson and Lindsay, 1977), as well as in the form of Zn complexes with nonprotein amino acids known as phytosiderophores (Tagaki et al., 1984) or phytometallophores (Welch, 1993). Concentration-dependent uptake of free Zn²⁺ ions has been shown to be saturable in several species, including maize (Mullins and Sommers, 1986), barley (Veltrup, 1978), and wheat (Chaudhry and Loneragan, 1972), suggesting that ionic uptake in grasses occurs via a carrier-mediated system. However, several of these studies have been criticized on the basis that excessively high (and physiologically unrealistic) Zn²⁺ concentrations were used (Kochian, 1993).

This study was undertaken to examine unidirectional Zn²⁺ influx and translocation to shoots in Zn-efficient bread wheat lines and Zn-inefficient durum wheat lines. Experiments were performed in the absence of added phytometallophores and results are presumed to represent influx of ionic Zn²⁺. Zn activities in the nanomolar range were used to more closely mimic free Zn²⁺ levels occurring naturally in soil solution. The results presented here indicate that a Zn-efficient bread wheat cultivar maintained higher rates of Zn uptake than a Zn-inefficient durum wheat cultivar, particularly at low (and physiologically relevant) solution Zn²⁺ activities.

	MATERIALS AND METHODS

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Seedling Growth

2

1

Uptake Experiments

In experiments measuring uptake from solutions containing free Zn²⁺ activities of less than 300 nM, EDTA was included in the uptake solution and free Zn²⁺ activities were calculated using the speciation program GEOCHEM-PC (Parker et al., 1994). In experiments measuring uptake at 2°C, uptake wells were packed in ice. To measure Zn²⁺ binding to root cell walls, roots were treated to disrupt and remove cellular contents. This was achieved by immersing roots in methanol:chloroform (2:1, v/v) for 3 d, followed by a rinse for 2 d in deionized water. Roots subjected to this treatment have been reported to yield a morphologically intact, lipid-free root cell wall preparation (DiTomaso et al., 1992). Unless noted otherwise, all experiments used a 20-min uptake period.

For pulse-labeled translocation experiments, seedlings were removed from wells following the 20-min uptake period in ⁶⁵Zn²⁺ and transferred to 1-L flasks containing nonradiolabeled ZnSO₄ in uptake solution. For continuously radiolabeled translocation experiments, seedlings were placed in 1-L flasks containing 4 µM ⁶⁵Zn²⁺. The uptake solution was replaced with fresh uptake solution containing 4 µM ⁶⁵Zn²⁺ after each harvest of a subset of the seedlings at specific time points. In all experiments, desorption was initiated at the end of the uptake period by replacing uptake solution with a 2°C desorption solution that contained 5 mM Mes-Tris (pH 6.0), 5 mM CaSO₄, 12.5 µM H₃BO₃, and 100 µM ZnSO₄. In translocation experiments, seedlings were transferred from flasks to uptake wells for desorption. After 15 min of desorption (with a change of desorption solution midway through the desorption period), seedlings were removed from uptake wells and placed on damp paper towels to remove excess solution from roots. Roots were excised, weighed, and analyzed for ⁶⁵Zn in an Auto-Gamma 5530 gamma counter (Packard, Meriden, CT).

	RESULTS

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Time-dependent Zn²⁺ accumulation in desorbed roots of bread and durum wheat varieties was linear for at least 90 min (Fig. 1). During this period, roots were immersed in a solution containing 4 µM Zn²⁺, and roots of the durum wheat cultivar accumulated less Zn²⁺ than roots of the bread wheat cultivar. Regression lines through the data points had r² values for bread and durum varieties of 0.984 and 0.987, respectively, and intercepted the y axis slightly above the origin. The amount of Zn²⁺ desorbed from roots of both wheat lines was dependent on the activity of Zn²⁺ in the uptake solution (Fig. 2). In the durum cultivar, after 60 min, approximately 60% of Zn²⁺ was desorbed from roots that had accumulated ⁶⁵Zn from a solution containing 4 µM Zn²⁺, whereas about 15% was removed from roots that had absorbed ⁶⁵Zn from a 66 nM Zn²⁺ solution. Desorption was rapid in both cases, with 76% and 60% of the total Zn²⁺, which was removed after 60 min of desorption, dissociating from roots within the first 2.5 min after incubation in 4 µM and 66 nM Zn²⁺, respectively. Results were similar for the bread wheat variety (not shown). In both wheat varieties the percentage of Zn²⁺ desorbed from roots increased as the activity of Zn²⁺ to which roots were exposed increased (Table I).

View larger version (19K): [in this window] [in a new window]   Figure 1. Time course of 65Zn2+ accumulation in bread and durum wheat seedlings. Roots were incubated in a solution containing 4 µM 65ZnSO4 and desorbed in a solution containing 100 µM nonradiolabeled Zn. Data points and bars represent means and SE values of four replicates. Error bars do not extend outside some data points. fr wt, Fresh weight.

View larger version (19K): [in this window] [in a new window]   Figure 2. Desorption of 65Zn2+ from intact durum wheat roots after a 20-min uptake period in solutions containing 66 nM or 4 µM ZnSO4. Data points and bars represent means and SE values of four replicates. fr wt, Fresh weight.

Concentration-dependent uptake kinetics for both wheat varieties were characterized by nonsaturating curves that became linear at Zn²⁺ activities greater than 20 µM (Fig. 3). These curves could be graphically dissected into saturable and linear components. Kinetic constants for the saturable components were derived by fitting a hyperbolic curve to the calculated saturable data points. Both K_m and V_max values were higher in the durum than in the bread wheat cultivar. At low Zn²⁺ activities, Zn²⁺ uptake rates were higher in the bread wheat variety (Fig. 4).

View larger version (27K): [in this window] [in a new window]   Figure 3. Concentration-dependent uptake of 65Zn2+ in intact bread and durum wheat roots. The data in each panel are from two separate experiments (Zn activities: 0.1-300 nM and 0.5-80 µM). Filled symbols depict total uptake. Dotted lines represent linear components derived from regression lines through the five highest concentration data points. Open symbols represent saturable components derived by subtracting the linear component from the total-uptake points. Data points and bars represent means and SE values of four replicates. Error bars do not extend outside some data points. fr wt, Fresh weight.

View larger version (20K): [in this window] [in a new window]   Figure 4. Concentration-dependent uptake of 65Zn2+ in intact bread and durum wheat roots. Uptake solutions contained 250 nM EDTA and varying concentrations of 65ZnSO4 (50-800 nM). Zn2+ activities shown on the x axis were calculated using the speciation program GEOCHEM-PC. Data points and bars represent means and SE values of four replicates. Error bars do not extend outside some data points. Inset, Low Zn2+ activity data points plotted on expanded axes. fr wt, Fresh weight.

Zn²⁺ uptake in both varieties was dramatically inhibited when roots were subjected to cold temperature or treated to remove cellular contents (Fig. 5). Zn²⁺ uptake in intact roots at 2°C was inhibited 70% to 85% in the bread wheat cultivar (Fig. 5) and 80% to 85% in the durum wheat cultivar (not shown) compared with uptake at 23°C. Methanol:chloroform-treated roots showed 70% to 80% (bread) and 60% to 80% (durum) reduction in uptake at 23°C compared with intact roots. At 2°C, Zn²⁺ uptake in methanol:chloroform-treated roots was reduced further in both varieties to about 85% inhibition. Zn²⁺ influx in both wheat varieties was also inhibited by Ca (Fig. 6). Increasing Ca activity caused greater inhibition of Zn²⁺ uptake, with a similar response in both cultivars.

View larger version (22K): [in this window] [in a new window]   Figure 5. Concentration-dependent uptake of 65Zn2+ at 23°C and 2°C in intact bread wheat roots and in bread wheat roots treated to remove cellular contents. The dotted line represents the linear component (Lin. comp.) derived from Figure 3A. Data points and bars represent means and SE values of four replicates. Error bars do not extend outside some data points. Meth/chl, Methanol:chloroform treated; fr wt, Fresh weight.

View larger version (18K): [in this window] [in a new window]   Figure 6. Uptake of 65Zn2+ in roots of bread and durum wheat seedlings. Uptake solutions contained 2 µM 65ZnSO4 and varying concentrations of Ca. Data points and bars represent means and SE values of four replicates. Error bars do not extend outside some data points. fr wt, Fresh weight.

In seedlings with roots exposed to ⁶⁵Zn²⁺ at three concentrations for 20 min and then transferred to solutions containing similar concentrations of unlabeled Zn²⁺, translocation of Zn²⁺ to shoots was time and concentration dependent in both wheat varieties (Fig. 7). Shoot Zn²⁺ concentration increased in both varieties during a 12-h period, with the greatest shoot concentrations measured in seedlings exposed to an uptake solution containing 4 µM Zn²⁺. When seedling roots were exposed continuously to 4 µM ⁶⁵Zn²⁺, shoot Zn²⁺ levels increased linearly for at least 24 h (Fig. 8). The bread wheat cultivar accumulated approximately twice as much Zn²⁺ as the durum wheat cultivar (Fig. 8). The partitioning of absorbed Zn within the plant (measured as shoot:root ⁶⁵Zn ratio) was similar in both varieties (Table II).

View larger version (23K): [in this window] [in a new window]   Figure 7. Time-dependent translocation of 65Zn from roots to shoots in intact seedlings of bread (A) and durum (B) wheat cultivars. Roots were immersed for 20 min in solutions containing different activities of 65Zn2+, and then transferred to solutions containing similar concentrations of nonradiolabeled ZnSO4. Data points and bars represent means and SE values of three replicates. Error bars do not extend outside some data points. fr wt, Fresh weight.

View larger version (16K): [in this window] [in a new window]   Figure 8. Time-dependent shoot translocation of 65Zn in intact seedlings of bread and durum wheat cultivars. Roots were immersed continuously in solutions containing 4 µM 65ZnSO4. Data points represent means and SE values of four replicates. Error bars do not extend outside some data points. fr wt, Fresh weight.

	DISCUSSION

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Zn²⁺ Binding

The linear kinetic component for Zn²⁺ influx (Fig. 3), which predominates at high Zn²⁺ activity in the uptake solution, can be interpreted as representing nonspecific Zn²⁺ binding to cell wall components that remains after desorption. Evidence for divalent cation binding to root cell walls that resists desorption when exposed to high cation concentration has been observed previously (DiTomaso et al., 1992; Hart et al., 1992; Lasat et al., 1996). As a means of estimating Zn²⁺ binding to wheat root cell walls, uptake was measured in intact roots exposed to low temperatures during the uptake period. Low temperature has been shown to inhibit uptake of Zn²⁺ in barley (Schmid et al., 1965) and sugarcane (Bowen, 1969).

The greatly reduced Zn²⁺ uptake in bread wheat roots under low-temperature conditions (Fig. 5) agrees with those earlier results. Furthermore, the plot of Zn²⁺ uptake under cold conditions displays a predominantly linear quality that is consistent with the linear components in Figure 3. For purposes of comparison, the linear component from Figure 3A has been replotted in Figure 5. The larger amount of root-associated Zn²⁺ in the low-temperature uptake experiment compared with the replotted linear component probably represents a low level of Zn²⁺ uptake under cold conditions in addition to nonspecific Zn²⁺ binding. The slightly saturable nature of the low-temperature uptake plot supports this interpretation.

Association of Zn²⁺ with roots treated to remove cellular contents must consist of nonspecific Zn²⁺ binding, and the plots in Figure 5 show much lower amounts of Zn²⁺ associated with roots subjected to this treatment. The higher amounts of Zn²⁺ binding in methanol:chloroform-treated roots compared with the replotted linear component (Fig. 5) may reflect greater accessibility to interior cell wall-binding sites exposed by the removal of the root symplasm. In addition, analysis of methanol:chloroform-treated roots (data not shown) revealed the presence of residual protein, which also could have contributed to higher levels of Zn²⁺ binding. Furthermore, the fresh weights of methanol:chloroform-treated roots were about 25% lower than those of intact roots because of the absence of intact cells, and this would lead to an overestimation of Zn²⁺ binding calculated on a per weight basis.

Taken together, the evidence from experiments with intact roots subjected to low temperature, as well as that from methanol:chloroform-treated roots, supports the interpretation that the linear components in Figure 3 represent nonspecific binding of Zn²⁺ to apoplasmic binding sites. As a consequence, the saturable components likely represent metabolically coupled transport of Zn²⁺ across the plasma membrane via a Zn²⁺ (or divalent cation) transporter.

The low level of cell wall binding seen in wheat roots in this work contrasts with the pronounced binding in barley roots reported previously (Schmid et al., 1965; Veltrup, 1978). The cause of this difference may be related to differences in root exposure to Zn²⁺ before the start of uptake experiments. In this work roots were grown in full nutrient solution containing Zn²⁺ (albeit at a low activity), whereas in the cited work, roots were grown without added Zn²⁺, either in low-salt medium (Schmid et al., 1965) or in nutrient solution (Veltrup, 1978). It is possible that in our experiments, growth in the presence of Zn²⁺ saturated Zn²⁺-binding sites in root cell walls and resulted in little additional binding of ⁶⁵Zn²⁺ in uptake experiments. Conversely, in the cited papers, Zn-deficient roots may have had binding sites with high affinity for Zn²⁺ occupied by Ca or other cations, which quickly exchanged with added Zn²⁺.

Zn²⁺ Uptake

The saturating curves for Zn²⁺ concentration-dependent uptake kinetics in both varieties (Fig. 3) is consistent with a carrier-mediated Zn²⁺ uptake system. Evidence for carrier-mediated Zn²⁺ transport has been reported in a variety of biological systems, including mammalian (Tacnet et al., 1990; Bobilya et al., 1992), fungal (Gadd et al., 1987; White and Gadd, 1987), and plant systems (Veltrup, 1978; Mullins and Sommers, 1986; Lasat et al., 1996). In the bread and durum wheat lines measured here, K_m values ranged between 2.3 and 3.9 µM (Fig. 3). Similar K_m values were reported for Zn uptake in roots of other gramineous crop plants, including barley (Veltrup, 1978), maize (Mullins and Sommers, 1986), and wheat (Chaudhry and Loneragan, 1972), as well as for fungal (White and Gadd, 1987; Budd, 1988; Sabie and Gadd, 1990) and animal (Bobilya et al., 1992) cells. The similar kinetic parameters for Zn²⁺ uptake among a wide variety of life-forms suggests conserved transport systems for Zn²⁺ or a common adaptation to similar ambient levels of this essential micronutrient.

The very low Zn²⁺ activities shown in Figure 4 were achieved by the use of the metal chelate buffer, EDTA. The chemical speciation program GEOCHEM PC was used to predict the free Zn²⁺ activities in the presence of varying total Zn²⁺ concentrations (50-800 nM) and a single EDTA concentration (250 nM). Experimental evidence from our laboratory clearly shows that in short-term experiments, Zn is taken up by wheat roots predominantly in the form of the free Zn²⁺ ion, and not as the Zn-EDTA complex (data not presented). Therefore, the free Zn²⁺ activities in the uptake solution in Figure 4 represent good estimates of the true free Zn²⁺ activities in solution in these experiments.

The significantly lower rates of Zn²⁺ uptake at low-solution Zn²⁺ activity in the durum wheat variety (Fig. 4) suggest that the Zn²⁺ uptake system is different from that in the bread wheat line. At higher Zn²⁺ concentrations, kinetic differences between the two wheat types were not as clearly resolved (Fig. 3). However, higher Zn accumulation (Fig. 1) and translocation to shoots (Fig. 8) over longer periods in the bread wheat cultivar were consistent with a higher capacity for net Zn uptake in bread wheat. Furthermore, the substantial difference in Zn²⁺ uptake rates between bread and durum wheats at low Zn²⁺ activities (Fig. 4) suggests that Zn efficiency may be related to the capacity for Zn²⁺ uptake from Zn-deficient soils.

Correlation between root Zn²⁺ uptake and Zn efficiency has been reported previously in studies of whole-plant net Zn²⁺ uptake rates in bread and durum wheat cultivars grown in Zn-deficient soils (Graham et al., 1992; Dong et al., 1995). Those studies showed lower Zn²⁺ uptake in Zn-inefficient durum wheat grown in long-term field and greenhouse pot experiments. However, a solution culture study that used chelate buffer to control Zn²⁺ activities at very low levels failed to establish a correlation between Zn efficiency and long-term (22 d) whole-plant net uptake rates (Rengel and Graham, 1995b).

It is important to note that the K_m values for both cultivars (2.3-3.9 µM) are higher than the soil-solution Zn²⁺ concentrations found in normal soils (1 nM-1 µM; Welch, 1995). In Zn-deficient soils, where the Zn-efficiency trait is expressed most clearly, Zn²⁺ activity in soil solution can be much lower, reaching a low to subnanomolar concentration range (Lindsay, 1991), far below the K_m values of the bread and durum wheat cultivars studied here. This suggests that short-term root uptake rates measured at Zn²⁺ concentrations much higher than those found in Zn-deficient soils may not be good predictors of Zn efficiency. The large difference in Zn²⁺ uptake rates of bread and durum cultivars measured at very low solution Zn²⁺ activities (Fig. 4) supports this view.

Responses of the Zn²⁺ transport system to certain external factors appear to be similar in both bread and durum wheat lines. Zn²⁺ uptake in both cultivars is inhibited dramatically by low temperature (Fig. 5) and by Ca (Fig. 6). The large inhibition of Zn²⁺ uptake at low temperature suggests a metabolic requirement for Zn²⁺ transport. As discussed by Kochian (1993), uptake of Zn²⁺ is likely to be a thermodynamically passive process, driven by the inwardly directed negative membrane potential across the plasmalemma, and low-temperature uptake inhibition is likely to result indirectly from a reduction in the membrane potential. Low-temperature-induced reduction in Zn²⁺ uptake was reported previously for sugarcane leaves (Bowen, 1969), barley roots (Schmid et al., 1965), and wheat roots (Chaudhry and Loneragan, 1972). The dramatic inhibition of Zn²⁺ uptake by Ca (Fig. 6) is also similar to findings from previous reports with rice (Giordano et al., 1974) and wheat seedlings (Chaudhry and Loneragan, 1972). In the latter study, transformation of uptake data into double-reciprocal plots revealed a noncompetitive interaction between Zn²⁺ and Ca²⁺, which suggested that Zn²⁺ and Ca²⁺ do not share a common transport mechanism. The parallel inhibitory response to low temperature and Ca in bread and durum wheat cultivars in this study implies that there are similarities in the Zn²⁺-transport systems of these two cultivars.

Zn²⁺ Translocation

In Figure 7, the decline in ⁶⁵Zn²⁺ accumulation in shoots with time should not be interpreted as saturation of shoots, but rather as the result of decreasing ⁶⁵Zn²⁺ specific activity caused by the replacement of radiolabeled solution by a solution containing nonradiolabeled Zn²⁺. When roots were exposed continuously to solutions containing Zn²⁺ at constant specific activity, translocation exhibited a linear time dependence for at least 24 h (Fig. 8). The larger amounts of Zn²⁺ translocated to shoots of bread wheat compared with durum wheat at a 4 µM Zn²⁺ root solution reflects the greater root uptake rate in the bread wheat variety. However, the similar shoot:root ⁶⁵Zn ratios (Table II) indicate that Zn partitioning was not different in the two varieties.

In summary, this work has provided evidence for carrier-mediated Zn²⁺ influx into the root symplasm in both durum and bread wheat varieties. It also demonstrates that Zn²⁺ uptake rates are lower in a durum wheat variety than in a bread wheat line, especially at low solution Zn²⁺ activities. Furthermore, the data show that Zn partitioning between root and shoot is similar in the two varieties. These results suggest that the rate of Zn uptake may be an important predictor of Zn efficiency. This information may be useful in breeding durum wheat lines that are more efficient in extracting Zn from soil solution. Alternatively, agronomic practices may be devised that increase Zn uptake in durum wheat by increasing the levels of available Zn in soils. Finally, the reduced Zn uptake rates in durum wheat measured in these experiments suggest that an investigation of the role of Zn-Cd interactions at the root surface may help in understanding Cd accumulation in low-Zn soils.

	FOOTNOTES

   Received February 9, 1998; accepted June 11, 1998.

	ACKNOWLEDGMENT

We thank the North Central Research Center (Minot, ND) for generously supplying the seeds of cvs Grandin and Renville.

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