Three Mechanisms for the Calcium Alleviation of Mineral Toxicities

Ca²⁺ Alleviation of Al³⁺ Toxicity

Ca²⁺ alleviates Al³⁺toxicity, but recent studies indicate that, ordinarily, the alleviation is not caused by the restoration of Al³⁺-inhibited Ca²⁺ uptake (Ryan et al., 1994) or by the restoration of Al³⁺-displaced cell-surface Ca²⁺ (Kinraide et al., 1994; Ryan et al., 1997). Ca²⁺ and other cations are ameliorative in the following order: Tris(ethylenediamine)cobalt³⁺ > Ca²⁺ ≈ Mg²⁺ ≈ Sr²⁺ > Na⁺ ≈ K⁺ (Kinraide and Parker, 1987; Kinraide et al., 1992, 1994; Ryan et al., 1994, 1997). Thus, it was concluded that Ca²⁺ and other cations relieve Al³⁺ toxicity solely by the displacement of Al³⁺ from the cell surface by electrostatic effects, i.e. by the reduction of cell-surface negativity and the consequent reduction of {Al³⁺}₀. Alleviation of toxicity by the electrostatic displacement of toxicant will be referred to as mechanism I. The use of {T}₀ instead of {T}_∞ in Equation 1 is an attempt to express that mechanism quantitatively.

If mechanism I is the sole means by which Ca²⁺relieves Al³⁺ toxicity, then Equation 1 will describe adequately root elongation, and an attempt to evaluate Equation 3 will lead to nonsignificant values fora ₂ and b ₂, as has been observed for all data published previously (analyses not presented). Nevertheless, Al³⁺ does displace Ca²⁺ from cell or PM surfaces (Reid et al., 1995;Ryan et al., 1997, Yermiyahu et al., 1997b), and Kinraide et al. (1994)predicted that under conditions of sufficiently low Ca²⁺, the addition of Al³⁺may induce a state of cell-surface Ca²⁺insufficiency.

To test that hypothesis, new experiments were performed to assess Al³⁺ intoxication in a Ca²⁺-Mg²⁺ replacement series in which [CaCl₂] + [MgCl₂] = 1 mm (Fig.1). This experimental design eliminated mechanism I as a variable because of the similar effects of Ca²⁺ and Mg²⁺ on ψ₀ (Kinraide et al., 1998). Figure 1 (top panel) demonstrates that [CaCl₂] > 0.1 mm([MgCl₂] < 0.9 mm) supported full root elongation in the absence of Al, but in the presence of 2 μm AlCl₃, [CaCl₂] > 0.2 mm was required. As [CaCl₂] declined below 0.2 mm,RRL _C also declined. This indicates that Al³⁺ caused a Ca²⁺insufficiency between 0.1 and 0.2 mmCaCl₂, and that Al³⁺aggravated the Ca²⁺ insufficiency already expressed in the controls below 0.1 mmCaCl₂. Alleviation of toxicity by Ca²⁺ restoration of toxicant-displaced Ca²⁺ will be referred to as mechanism II. The use of {Ca²⁺}₀ instead of {Ca²⁺}_∞ in Equation 2 is an attempt to express that mechanism. Mechanism II is related to the Ca²⁺-displacement hypothesis for toxicity, which states that a cation is toxic because it displaces Ca²⁺ from the cell surface.

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Fig. 1.

Root elongation in response to CaCl₂, MgCl₂, and AlCl₃ in the rooting medium. [CaCl₂] + [MgCl₂] = 1 mm and pH = 4.6. RRL _C is the root length in Al solutions relative to Al-free solutions with similar CaCl₂and MgCl₂. The data were compiled from six experiments, with each point on the graph based on one to six values. Each of these values was based on the measured lengths of 10 roots, as described inMethods.

If mechanisms I and II both operate in the alleviation of Al³⁺ toxicity, then Equation 3 (not incorporating Eq. 4) should be appropriate. The experiments presented in Figure 1were pooled with the new experiment presented in TableI. Because the pH was only moderately low in these experiments (4.6) and the sum of the divalent cations was moderately high (1 mm), the roots were under no H⁺ stress (Kinraide et al., 1992; present study). Regression analysis according to Equation 3 yielded the results presented for Al³⁺ in TableII. When Equation 4 was incorporated into Equation 3, a nonsignificant value for a ₄was obtained. Figure 2 presents model-generated curves for RRL _max and 1 − 1/exp[(a ₂{Ca²⁺}₀)^{b₂] based on the coefficients in Table II. Because all solutions for a given toxicant are electrostatically similar, mechanism I does not influence the trends in Figure 2.}

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Fig. 2.

Calculated root responses to CaCl₂, MgCl₂, and toxicants in the rooting medium. [CaCl₂] + [MgCl₂] = 1 mm and pH = 4.5, except where H⁺ was the designated toxicant. Toxicant levels (AlCl₃ = 3.06 μm, pH = 4.12, and NaCl = 132.2 mm) were chosen so thatRRL _max = 50 at [CaCl₂]/([CaCl₂] + [MgCl₂]) = 0.5. ψ₀ was −14, −15, and −10 mV for the added Al³⁺, H⁺, and Na⁺, respectively. The constancy of ψ₀ for a toxicant means that electrostatic displacement of toxicant by Ca²⁺ (mechanism I) played no role in the computed trends. A ₂= 1 − 1/exp[(a ₂{Ca²⁺}₀)^{b₂] and is an expression of mechanism II alleviation. The continued increase in} RRL _max for Na⁺ and H⁺ in the top panel signifies the action of mechanism III.

Ca²⁺ Alleviation of H⁺ Toxicity

Ca²⁺ alleviates H⁺toxicity, and mechanism I appears to operate because toxicity is also alleviated by Na⁺, Mg²⁺, and Tris(ethylenediamine)cobalt³⁺ and other cations whose effectiveness increases with increasing charge (Kinraide et al., 1992). Nevertheless, the pattern of Ca²⁺alleviation is different from that of the Ca²⁺alleviation of Al³⁺ toxicity because Ca²⁺ is a more effective ameliorant than Mg²⁺, even at relatively high [Ca²⁺]_∞, where mechanism II would not be expected to operate. In Ca²⁺-Mg²⁺ replacement experiments, RRL _C continued to increase with increasing [Ca²⁺]_∞(Kinraide et al., 1994). Growth experiments with H⁺ indicate that the effectiveness of the toxicant may be reduced by Ca²⁺ by mechanisms beyond those already described. For that reason, we introduce a third class of mechanisms: the alleviation of toxicity by Ca²⁺ by one or more mechanisms other than I and II will be referred to asmechanism III. The incorporation of Equation 4 into Equation3 is an attempt to express these mechanisms.

If mechanism III does operate, then Equation 3 (not incorporating Eq.4) will be incomplete, and incorporation of Equation 4 into Equation 3may improve the description of root elongation. These ideas were tested by pooling results from several published experiments (Kinraide et al., 1994) with results from new experiments such as those presented in Table III. The data were evaluated in terms of Equation 3 (incorporating Eq. 4). The results of the regression analysis are presented in Table II and Figure 2, where the effects of mechanism III on H⁺ toxicity can be seen in the continued increase of RRL _maxfor H⁺.

Ca²⁺ Alleviation of Na⁺ Toxicity

Ca²⁺ alleviates Na⁺toxicity, but a preliminary inspection of the data indicates that Ca²⁺ alleviation is not dominated by mechanism I to the extent that it is in Al³⁺ toxicity or even H⁺ toxicity. The diagnostic feature of mechanism I alleviation is that cations other than Ca²⁺ are effective ameliorants. Mg²⁺ and Sr²⁺ do reduce Na⁺toxicity, but the effects are weak compared with the effect of Ca²⁺ (Fig. 3). Furthermore, high Na⁺ can impose an osmotic stress that Ca²⁺ is unable to relieve.

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Fig. 3.

Effects of NaCl, CaCl₂, and MgCl₂ on root elongation. A factorial array of 27 solutions contained 80, 105, or 130 mm NaCl, 0.15, 0.30, or 0.45 mm CaCl₂, and 0, 1, or 2 mmMgCl₂ (all at pH 5.0). Areas of circles are proportional to [CaCl₂]. The data are from a single experiment representative of several similar but not identical Na⁺-Ca²⁺-Mg²⁺ experiments.

Data from several experiments were pooled (including the data from Fig.3, which were published previously [Kinraide, 1994]), and data from some new experiments in which [Na⁺]_∞ ranged from 0 to 200 mm were also used. In addition, data were examined from an experiment in which mannitol ranged from 0 to 400 mm. Mannitol concentrations < 250 mm were not inhibitory, and Ca²⁺ could relieve Na⁺toxicity completely when the [NaCl] was <135 mm, indicating that osmotic stress was not a factor in root elongation in solutions with an osmolarity <250 mm. Consequently, only data from solutions with [NaCl] < 135 mm were retained in the pool. Regression analysis of these data yielded the results presented in Table II and Figure 2, where strong mechanism III effects can be seen in the steep and continuous increase ofRRL _max for Na⁺.

The coefficient a ₁ can be defined to include a Mg²⁺ term. If the equation:a1=a3/(a4{Ca2+}0+a7{Mg2+}0+1) Equation 5is incorporated into Equation 3, regression analysis yields six statistically significant coefficients (Table II, last line). Use of Equation 5 for the analysis of Al³⁺ or H⁺ toxicity resulted in nonsignificant values for coefficients.

A Reevaluation of a H⁺-Toxicity Study in an Attempt to Discern Mechanism II

The analytical techniques described above were also applied to several published studies in which a role for mechanism II was asserted or implied. In most cases mechanism I accounted for most of the alleviation, and mechanism II could not be confirmed. The following example illustrates some important points of experimental design and analysis.

In a study of H⁺ toxicity, Yan et al. (1992)concluded that the inhibition of corn root elongation was likely to have been a consequence of H⁺ influx inadequately compensated for by H⁺ secretion, resulting in the acidification of the cytoplasm. H⁺ displacement of Ca²⁺ was thought likely to have enhanced H⁺ influx, an effect that could be offset by the addition of Ca²⁺ to the medium.

To determine whether inhibition of corn-root growth by H⁺ was demonstrably a function of Ca²⁺ displacement, data were pooled from the figures and tables of Yan et al. (1992). Growth was scaled toRRL _max, {H⁺}₀ and {Ca²⁺}₀ were computed, and RRL _max was plotted against the surface ion activities in Figure 4. Growth appeared to be inhibited by {H⁺}₀ and enhanced by {Ca²⁺}₀, but in this study, as in most others, {H⁺}₀ and {Ca²⁺}₀ were intercorrelated (Fig. 4c), so an effect of Ca²⁺other than the mechanism I effect is difficult to discern. Analysis in terms of Equation 3 (incorporating Eq. 4) yielded statistically significant values for a ₁ (4.38),b ₁ (3.00), anda ₂ (5.68). b ₂and a ₄ could not be evaluated so they were deleted (effectively setting b ₂ equal to 1). Apparently, the correlation of {H⁺}₀ with {Ca²⁺}₀ was not strong enough to prevent the evaluation of both a ₁and a ₂, so one may conclude, in agreement with Yan et al. (1992), that H⁺-induced Ca²⁺ displacement accounts for some of the intoxication. Nevertheless, the data set is inadequate for extensive analyses, mainly because of {H⁺}₀ and {Ca²⁺}₀intercorrelations.

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Fig. 4.

Growth of corn seedling roots in response to H⁺ and Ca²⁺. Data were taken from Yan et al. (1992). Growth measurements were scaled toRRL _max, and activities in the medium and at the PM surface were computed as described elsewhere in the present study. Areas of circles are proportional to {Ca²⁺}₀ or {H⁺}₀, as indicated.