Copper Uptake by Excised Roots III. Effect of Manganese on Copper Uptake

STEPHEN J. HARRISON!), NICHOLAS W. LEPP!) and DAVID A. PHIPPS2)

1) Dept. of Biology and 2) Dept. of Chemistry and Biochemistry, Liverpool Polytechnic, Byrom Street, Liverpool L3 3AF, U.K.

Received September 27,1982' Accepted December 9,1982

Summary

The effect of manganese on uptake of copper by excised Hordeum roots was examined. Copper uptake was reduced in the presence of manganese; a similar situation was evident for manganese uptake. The pattern of interaction between the two elements was highly suggestive of competitive inhibition, with copper and manganese, showing a common uptake site. The rates of transport of the two elements was not similar at the same molar concentration; copper showed an affinity for the uptake site 3.7 times greater than that of manganese.

Key words: Hordeum vulgare, excised roots, manganese, copper uptake.

Introduction

Evidence has accumulated over the last decade which indicates that copper (Veltrup 1976; Harrison et a1. 1979; Bowen 1981) and other essential nutrient ele­ments (Marquenie-van der Werff and Ernst 1979; Nissen 1973; Hassan and Tang Van Hai 1976) are accumulated in plant roots by a mechanism which follows multiphasic kinetics. Nissen (1973) has interpreted such kinetics as a product of a single carrier mechanism, which undergoes «all or none» transitions at specific concentrations of transported substrate. This produces abrupt changes in the values of the kinetic para­meters describing uptake. A mechanism of this type has been shown to be modified by the presence of ions with similar charge and ionic radius to those transported. This modification may take one of two forms: (a) Inhibition of uptake as a result of competition for uptake sites (Bowen 1981). (b) More complex alteration of uptake rate, as a consequence of interference with a

«transition site» regulating the operational phase of uptake (Vange et a1. 1974; Nissen 1980).

The studies described below were conducted to investigate interactions between copper and manganese during uptake of both ions, by excised roots, and to further examine the nature of such processes as and when they were found to occur.

Materials and Methods Excised roots of barley (Hordeum vulgare L. c.v. Aramina) were employed throughout this

study. Methods were similar to those described in Harrison et al. (1978). Briefly, roots were

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286 STEPHEN J. HARRISON, NICHOLAS W. LEPP and DAVID A. PHIPPS

obtained by sowing seeds on stockinette mesh which was placed in contact with the surface of a solution of 0.5 mM CaCh in 10 litre plastic bowls. The solution was aerated and the seedlings maintained under complete darkness until excision on the 7th day after sowing. Root samples were washed and retained until required in 0.5 mM CaCh, then loaded into «swimfeeders», the use of which was described previously (Harrison et al. 1978), to facilitate transfer to the relevant uptake solution, which also contained CaCh at this concentration. The time from excision to transfer to uptake solution was less than two h. At the end of the uptake period roots were rinsed for 10 s in each of two changes of de-ionised water and then transferred to a cooled (SoC) solution of 5 mM ,Pb(N03h, to desorb copper from the Donnan Free Space (Harrison et al. 1979). Roots were removed after 1 h, blotted and oven dried to constant weight at 80°C. Samples were then digested under pressure with 1 ml of HN03 in scintillation vials placed in a water bath at 70 0C. The samples were finally made to 10 ml volume and analysed for Cu using an IL 151 atomic adsorption spectrophotometer.

Results and Discussion

Fig. 1 shows the results of an experiment in which excised roots were exposed for thirty min to CUS04 solutions at a range of concentrations, with or without added Mn2+ as MnCh at a concentration of 182 /-tM. The data are presented in the form [S]/V against [S], where [S] is the applied concentration and V the rate of transport. Previous work has shown that this will yield linear plots over this concentration

12 +Mn

11 -Mn

10

M V 9

0

8

7 0

50 70 90 110 130

eu /JM

Fig. 1: The relationship of copper uptake rate V, and applied concentration [S], with or without additional Mn2+ as 182/LM MnCh . • --. + Mn, 0--0 - Mn. The lines were drawn from the kinetic constants of Table 1.

range due to uptake following simple Michaelis-Menten Kinetics. Although uptake of Cu has been described as multiphasic, in this instance concentration values lie within a single phase of the uptake isotherm (Harrison et aI. 1979). There is a clear difference in the uptake of Cu2+ with and without added Mn2+. In the presence of Mn2+ the value of [S]/V is increased relative to the value obtained for roots without added Mn2+, because of a decrease in V, the rate of copper uptake. The lines drawn on the

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Copper uptake by roots 287

Fig. were calculated using an iterative computer method based on that of Cleland (1963), which fits the best curve to the equation:

I V =I rVmaxX[5]] og og tKm + [5]

Where V is uptake velocity, [S] transported ion concentration, Vmax the maximum rate of ion transport and Km the concentration at which half maximal transport is attained.

Table 1: Kinetic constants and 95 % confidence limits for the inhibition of copper uptake from Cu504 solutions over the range of concentrations shown in figure 1, in the presence of Mn2+ as 182/lM MnCh.

-Mn 103.40±42.64 +Mn 170.23±54.95

*) dry weight

21.42±4.82 24.83±5.17

The values of Km and Vmax returned by this method are provided in Table 1. It would appear that the reduction of Cu2+ uptake in the presence of Mn2+ is a result of an increase in the value of Km, which is a measure of apparent affinity of Cu2

+ for the uptake sites, an increase in Km representing a reduction in affinity. Such a pattern of inhibition is characteristic of competition for uptake sites (Cornish-Bowden 1979) where:

Km (apparent) = Km (1 + I1KJ)

and I is inhibitor concentration and KJ the apparent dissociation constant for the reac­tion of inhibitor with uptake site.

To gain further information on the Cu2+ IMn2+ interaction the Mn2+ absorption

0·24 •

0·12

50 70 90 110 130

eu )JM

Fig. 2: Relationship between the uptake of Mn from a solution of 182/lM MnCh, plotted as the reciprocal of uptake rate, and the concentration of Cu2+ present in the uptake solution. Regres­sion line: y = 0.000989 x + 0.1112.

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288 STEPHEN J. HARRISON, NICHOLAS W. LEPP and DAVID A. PHIPPS

Fig. 3: The ratio of the rates of Mn2+ and Cu2+ uptake plotted against the ratio of the concentration of these as supplied. Regres­sion line: y = 3.7522 x - 0.147.

2-6

2·2

1·0

0·3 0·4 0·5

IsICu IsIMn

0·6

•

0·7

of the roots was also measured for those treatments receiving added Mn2+. These data are shown in Fig. 2. The data are plotted lIv against [S] and show that as Cu2

+

concentration increased l/v for Mn2+ uptake increases, indicating a decrease in Mn2+ uptake velocity, thus confirming a reciprocal pattern of competition for these two ions.

Plotting the ratios of uptake for the two metals against the ratio of applied metal concentrations provided additional information relating to the nature of the competi­tion and relative affinities of the two metals for the uptake sites. This process is illustrated in Fig. 3 which shows a linear relationship between the two variables. From this it is possible to make the tentative conclusion that transport of these two ions is by a common mechanism. That is the competitive affects are a result of a single shared uptake system rather than two or more mechanisms, not all of which are shared. This conclusion is based on the linearity of the plot, as the additional influence of an uninhibited mechanism for either Mn2+ or Cu2+ would produce curvature in these data. Further, from the slope of the line we can estimate that the affinity of Cu2+ for the site is 3.7 times that of Mn.

These findings contrast with those of Bowen (1969, 1981) who has reported no interaction between Cu2+ and Mn2+ for uptake into both barley roots and sugar cane leaf tissue. He concluded that Cu2+ and Zn2+ share a common uptake mechanism, and show competitive inhibition, whilst Mn2

+ is absorbed by a separate mechanism. This suggests that these mechanisms are variable within the species Hordeum vulgare, a deduction supported by the evidence of Veltrup (1977, 1978), who reported that for his material and experimental conditions there was no influence of Zn2+ on Cu2+

uptake, although conversely Cu2+ does affect Zn2+ uptake but not competitively. The nature of the interaction between Cu2+ and Mn2+ reported here is distinct

from that expected if the metals modified a «transition» site separate from the uptake site but controlling it, as described by Veltrup for Zn and Cu (1977, 1978) and by Vange et al. (1974) and Nissen (1980) for other ions. Such a postulated mechanism is

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Copper uptake by roots 289

characterised by abrupt responses to transport analogues as opposed to the competi­tive phenomenon reported here. These findings may be characteristic of the phase of the uptake isotherm in which the experiments were conducted and do not preclude other interactions between Cu2+ and Mn2+ at concentrations beyond the ranges studied here.

References

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- Kinetics of active uptake of boron, zinc, copper and manganese in barley and sugarcane. J. Plant Nutrition 3,215-233 (1981).

CLELAND, W. W.: Computer programmes for processing enzyme kinetic data. Nature 198, 463-465 (1963).

CORNISH-BoWDEN, A.: Fundamentals of enzyme kinetics. Butterworths, London, 1979. HARRISON, S. J., N. W. LEPP, and D. A. PHIPPS: Uptake of copper by excised roots. I. A modified

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MARQUENIE-VAN DER WERFF, M. and W. H. O. ERNST: Kinetics of copper and zinc uptake by leaves and roots of an aquatic plant, Elodea nuttallii. Z. Pflanzenphysiol. 92,1-10 (1979).

NISSEN, P.: Multiphasic uptake in plants. II. Mineral cations, chloride and boric acid. Physiol. Plant. 29, 298-354 (1973).

- Multiphasic uptake of potassium by barley roots of low and high potassium content: sep­arate sites for uptake and transitions. Physiol. Plant. 48, 193-200 (1980).

V ANGE, M. S., K. HOLMERN, and P. NISSEN: Multiphasic uptake of sulphate by barley roots. I. Effects of analogues, phosphate and pH. Physiol. Plant. 31,292-301 (1974).

VELTIlUP, W.: Concentration dependent uptake of copper by barley roots. Physiol. Plant. 36, 217-220 (1976).

- The uptake of copper in the presence of zinc. Z. Pflanzenphysiol. 83, 201-206 (1977). - Characteristics of zinc uptake by barley roots. Physiol. Plant. 42, 190-194 (1978).

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