Physiol, Plant, 49: 383-387, 1980 EFFECT OF CALCIUM ON SULFATE UPTAKE 383

Effect of calcium on sulfate uptake by barley roots

By

EGIL SKJELBREID and PER NISSEN^

Department of Microbiology and Plant Physiology,University of Bergen, Allegaten 70,

N-5014 Bergen, Norway

(Received 20 December, 1979; revised 27 March, 1980)

Abstract

Sulfate uptake by excised roots of barley (Hordeum vulgare L.)was maximal in the presence of about 3xlO~^M CaCK, Kineticstudies contraindicate a stoichiometric binding of calcium to thecarrier for sulfate, in contrast to findings of Cuppoletti and Segel(Biochemistry 14: 4712^718, 1975) for the filamentous fungusPenicillium notatum. In barley, calcium affects the K ^ but not theVmax for sulfate uptake, presumably by altering the conformationand, thereby, the affinity of the carrier. Calcium also affects thetransition site for sulfate uptake.

Key words: Sulfate uptake - calcium - Hordeum - uptake site -transition site.

Introduction

Calcium has long been known to be essential for thestructural and functional integrity of plant membranes (cf.Epstein 1972), Consistent with this is the common find-ing, first made by Viets (1944), that a certain optimallevel of calcium is required for maximal uptake of nu-trient ions. Calcium acts presumably by maintaining themembrane with its proteins and lipids in a properphysico-chemical state. The effects may be complex, andnot always positive, as found for uptake in various plants(refs, in Leonard et al. 1975), The precise mechanism(s)of action of this divalent cation remain(s), however, to beelucidated.

In a study of sulfate transport by the filamentous fungusPenicillitim notatum, Cuppoletti and Segel (1975) foundby kinetic analysis that a proton and a calcium ion bind tothe carrier before sulfate. In the present study we haveasked whether a similar mechanism may be involved insulfate uptake by higher plants. The results contraindicateany binding of calcium to the sulfate carrier in barley

To whom correspondence be addressed.

roots, i.e. the role of calcium is less direct than in thefungal system.

Materials and methods

Barley seeds {Hordeum vulgare L, cv, Herta) were soakedin aerated H2O for about 24 h and then grown in the darkat about 20°C in 45-liter aquaria containing aerated5x \()~*M CaCl2. Roots (average length about 8 cm) wereexcised from 7-day-old plants and rinsed several times indistilled H2O,

Uptake experiments were done with about 2,5 g rootsin 100 ml of solution on a gyratory shaker at about 22°C,The uptake solutions contained '"^S-labeled K2SO4 (about3x10* dps/|amol) and CaCl2 of indicated concentrations.The 20 min uptake period was followed by a 5 min washin cold (about 8°C) H2O, Root samples (1,0 g fresh wt,)were assayed for radioactivity by hquid scintillationcounting.

Maximal depletion of sulfate during uptake will occurfor 10-''/W K2SO4 and 3 x 1 0 " ^ CaCl2 and can from Fig-ure 1 and the root-to-solution ratio be calculated to beabout 25%, The average sulfate concentration will in thiscase be 12-13% lower than the initial concentration. Theerror will, however, be substantially less for higher sulfateconcentrations and for nonoptimal calcium concentra-tions. The decrease in free calcium due to binding anduptake can similarly be estimated to be about 10 and 5%for 1()~*M and 10"*M CaCl2, respectively. The decrease isnegligible for high calcium concentrations. The uptakesolutions were unbuffered and ranged initially around pH6 (somewhat higher for the low calcium concentrations inTable 1, somewhat lower for the intermediate and highconcentrations). The pH remained relatively unchangedfor the low and intermediate concentrations but was atthe end of the experiment about one unit lower for thehigh calcium concentrations.

0031-9317/80/080383-05 $03,00/0 © Physiologia Plantarum

384 EGIL SKJELBREID AND PER NISSEN Physiol, Plant, 49. 1980

ResultsUptake of sulfate from 10-*M or 2x Kj- M solutions was3-6 fold higher in the presence of 3xlO--^M CaCl2than atvery low calcium concentrations (Figure 1), Supraoptimalconcentrations were highly inhibitory, uptake being re-duced to one-tenth at 3xlO"'M CaCi2. These resultsagree well with the more comprehensive findings ofHolmem et al. (1974),

Sulfate uptake from a 2x10^ M solution at subopti-mal calcium concentrations is shown in Figure 2, Thedouble reciprocal plot for barley roots is clearly cur-vilinear, in contrast to the linear plots for Penicilliumnotatum (Figure 2, insert). Experiments with other sulfate

Figure 1, Effect of calcium on uptake of sulfate by barley roots.Average SE: 8,8% (n = 4),

l/fca'*],

Figure 2, Double reciprocal plot (l/v vs. ]/[Ca^*D for uptake ofsulfate by barley roots in the presence of lO'^M - 5y~10~*MCaCU Average' SE: 4,07o (n = 3), Insert: Double reciprocalplots for uptake of sulfate by Penicillium notatum, simplifiedfrom Fig, 5 in Cuppoletti and Segel (1975),

Figure 3, Double reciprocal plots (l/v vs. 1/[SO^ ])/"'• uptake ofsulfate by barley roots in the presence of various suboptimal con-centrations of CaCl2. Average SE: 9,7% (n = 5),

l/v

10^

Figure 4, Double reciprocal plots (l/v vs. l/[SOl ]) for uptake ofsulfate by barley roots in the presence of various supraoptimalconcentrations of CaCl2. Average SE: 8,0% (n = 5),

concentrations did also give curvilinear plots. This indi-cates that there is no direct interaction between calciumand sulfate in the plant, cf. Discussion.

The concentration-dependence of sulfate uptake atvarious sub- and supraoptimal calcium concentrations is

Physiol, Plant, 49, 1980 EFFECT OF CALCIUM ON SULFATE UPTAKE 385

Table I, Kinetic constants for the experiments in Figures 3 and 4.Values for Vn, ,, in nmol/g fresh wt • 20 min. Values for K , intxM. For calculations, see Nissen (1977), 95% confidence inter-vals indicated.

0,03-

Calciumconcentration

10-*3x10-*

1 0 ^3x10^

10-2

3x10-^10-'

3x10-'

* max

78±2278+ 864± 785± 6

93±13105±10150±6970±30

36±1123± 311± 19± 1

10± 218± 261±30

113±50

shown in Figures 3 and 4, respectively. The sulfate con-centrations {IQ'^M - 10-^M) are all in the range of phase1 of the mechanism for sulfate uptake (Nissen 1971), andthe double reciprocal plots are, as expected, linear for allcalcium concentrations.

The kinetic constants for the plots in Figures 3 and 4are given in Table 1. The V^a^ values were identical orsimilar for suboptimal calcium concentrations and some-what variable but roughly similar for supraoptimal con-centrations. The Kp, values increased markedly, i.e. theaffinity (K^,"') decreased, as the calcium concentrationwas made increasingly sub- or supraoptimal.

In the presence of 10-^M, lO^'M or lO-^M CaCl2, sul-fate uptake in the range KJ-^ M - 2,5xlO^M was, asexpected, bi- or triphasic (Figure 5); cf, also Vange et al.(1974) and Holmerne/a/. (1974). The transition betweenphases 2 and 3 (arrows) occurred at different externalsulfate concentrations for the different calcium concen-trations (Table 2). For both phases the affinities de-creased (Km increased) as the calcium concentration wasmade increasingly suboptimal, in agreement with the re-sults in Table 1. In another experiment (not shown), thetransition between phases 2 and 3 occurred at about10-^M sulfate for lO- A/ CaCl2 and at about b.2>x\i)-^Msulfate for lO^M and \0-^

l/v

0,02-

EOCJ

S

0,01 -

Figure 5, Double reciprocal plots (l/v vs. l/[SOl J) for uptake ofsulfate by barley roots in the presence of various suboptimal con-centrations of CaCl2. Range of experiment H)~^M - 2.5y~\()~*M,range shown I,6x lO-'M - 2,5xlO"^A/, Transitions indicated byarrows. Average SE: 11,9% (n = 5),

Discussion

On the basis of a comprehensive kinetic analysis of theactive transport of sulfate by Penicillium notatum, Cup-poletti and Segel (1975) concluded that a proton and acalcium ion bind in random order to the carrier beforesulfate. The order of binding of calcium and sulfate wasdetermined by measuring sulfate transport for variousexternal concentrations of sulfate and calcium. The lineardouble reciprocal plots obtained with calcium as the var-ied substrate (insert in Figure 2) and replots of these dataare consistent with a reaction in which calcium adds to thecarrier before sulfate; cf, also Segel (1975),

The finding of curvilinear rather than linear double re-ciprocal plots for barley roots {e.g. Figure 2) contraindi-cates a similar binding of calcium to the sulfate carrier inthe plant. In the case of the roots, sulfate uptake willoccur, albeit at a reduced rate, in the absence of calcium.

Table 2, Kinetic constants for the experiment in Figure 5. Values for V^^,, in nmol/g fresh wt • 20 min. Values for K^ in \iM.Significance of fit to adjacent phases (tested against a single phase): " " - not significant {p > 0,05), * - 0,01 <p S 0,05, See Nissen(1977) for analytical procedure, 95% confidence intervals indicated.

Calcium concentration(M)

Phase Transition(M)

10-* 2323

23

224± 50555±136265± 214971182

205 ± 41395± 45

88± 27330±11068± 9

210±14027± 990± 20

6,3x10-'(chosen)*

10- (chosen)*

4,0x10-'(chosen)"'

386 EGIL SKJELBREID AND PER NISSEN Physiol, Plant, 49, 1980

In the fungal system on the other hand, the double recip-rocal plots (insert in Figure 2) show that there will be notransport in the absence of calcium.

It has been suggested that diffusion through the freespace of the root can limit the uptake of phosphate at lowionic strength (Pettersson 1975), It seems unlikely, how-ever, that diffusion was rate limiting in the present ex-periments even at 10-''M, the lowest concentration ofsulfate. Rates for uptake of sulfate are less than one-tenthof those for phosphate (Vange et al. 1974), and there is noindication in the present data of diffusion being rate-limiting. In all probability the observed effects of calciumrelate, therefore, to uptake across the plasmalemmarather than to diffusion in the free space. It also seemsthat any change in pH or decrease in the concentration ofsulfate or calcium during the experiment (see Materialsand methods) will only affect the results in a quantitativeand relatively minor way.

For sulfate uptake by yeast, Roomans et al. (1979) haverecently proposed a model in which the stimulation bydivalent cations is due to changes in the surface potential.The results are similar to those in the present paper inthat the optimal cation concentration was about 4x lO-'A/and that only K , was affected. The results differ fromthose for barlc\ in that there was only a single phase up to5x lO^A/ sulfate, the highest concentration used. Furthercomparisons are precluded or difficult because of the li-mited scope of the present study.

The molecular mechanisms underlying the effects ofcalcium on membrane transport in higher plants remainunknown, but it has been suggested that calcium maycause conformational changes in membrane lipids {e.g.Kreutz 1972), Such changes may in turn alter the struc-ture and functioning of carrier proteins or other entitiesinvolved in transport. From kinetic studies it seems clearthat calcium affects both the uptake and the transition sitefor sulfate, [See Vange et al. (1974) for the existence ofseparate uptake and transition sites,] Calcium apparentlyalters the uptake site, either directly or via surroundinglipids, and renders its affinity for sulfate maximal at anoptimal concentration of the divalent cation. At this con-centration, calcium may be said to "tighten" the uptakesite; cf, the discussion of "loose" and "tight" structuresfor multiphasic ion uptake (Nissen 1980),

The kinetic mechanism by which calcium stimulatessulfate uptake in roots remains to be determined. In thecase of a non-essential activator, the plots for l/v versusl/[SOi-] should have intersected to the left of the l/v axis(Segel 1975) rather than at the axis as in the presentexperiments (Figure 3), Why high calcium concentrationsmake the uptake site less "tight", as indicated by theincrease in Kp, (Table 1), and thus depress uptake is notknown. The decrease in the pH of the uptake solution(see Materials and methods) suggests that the plas-maletnma may have been damaged, but this is contraindi-cated by the precise kinetics in Figure 4, The decreaseshould in itself increase uptake by bringing the pH closer

to the optimum of 4, cf, Vange et al. (1974), In contrast toits effect on K , calcium had little or no effect on V^ax atsuboptimal concentrations and only comparatively littleeffect at supraoptimal concentrations.

In addition to its effect on Kn, calcium altered the con-centrations of sulfate needed to bring about transitions.This shift in transitions may not be very marked butagrees with previous findings (Holmern et al. 1974),Furthermore, phase transitions did not occur at low sul-fate concentrations in the absence of calcium or otherdivalent cations (Holmern et al. 1974),

Sulfate transport in filamentous fungi obeys simpleMichaelis-Menten kinetics over a wide range of extracel-lular sulfate concentrations, with no indications of anytransitions or discontinuities (Bradfield et al. 1970), Thisis in striking contrast to the multiphasic and discontinuousisotherms found for uptake of sulfate (Nissen 1971, 1973,Vange et al. 1974, Holmern et al. 1974, Panopoulose/ al.1975, Shargool and Ngo 1975) and other ions (Nissen1974) in higher plants. Goring and coworkers (Grunwaldtet al. 1979 and references therein) have proposed that thecomplex kinetics of ion uptake in higher plants can simplyresult from the plant tissues being multilayered. It seemsevident, however, that multiphasic kinetics are indeed aproperty of the plasmalemma of the individual cell. Thekinetics are multiphasic not only in multilayered roots butalso in tissues with only two or a few cell layers, e.g. leavesof Elodea densa (Nissen 1973) and thin leaf slices of bar-ley (Nissen 1971), respectively. Unfortunately, the rangeof sulfate concentrations was insufficiently wide to ascer-tain the kinetics of uptake in suspension cultured tobaccocells (Smith 1976), The finding by Mettler and Leonard(1979) of similar, and complex, kinetics of ion uptake intobacco cells and protoplasts from these shows, however,that complex kinetics are neither an artifact associatedwith multicellular tissues nor a result of ionic interactionwith cell walls. It is probably also relevant that the kine-tics of K "^-stimulation of an ATPase from a plasmalem-ma-enriched root fraction are multiphasic with transitionsevident as sharp breaks or jumps (Nissen 1977,Havarstein and Nissen 1980),

Various properties of uptake mechanisms have beenshown to vary along the length of the roots {e.g. Eshel andWaisel 1973, Marschner and Richter 1973, Cacco et al.1977), The multiphasic patterns are, nevertheless, notappreciably blurred, and variations along roots probablyreflect quantitative rather than qualitative changes (Nis-sen 1974), Multiphasic isotherms for nutrient concentra-tions are even obtained after growth of plants for weeksor months (Nissen et al. 1980), again indicating that theisotherms reflect a fundamental property of the plas-malemma. The differential kinetics of sulfate transport infungi and higher plants appear, therefore, to reflect a dif-ference in membrane structure rather than merely thegreater complexity of the plant tissue. This difference mayhopefully facilitate the study of the molecular basis ofconcentration-dependent phase transitions.

Physiol, Plant, 49, 1980 EFFECT OF CALCIUM ON SULFATE UPTAKE 387

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Cacco, G,, Saccomani, M, & Ferrari, G, 1977, Development ofsulfate uptake capacity and ATP-sulfurylase activity duringroot elongation in maize, —Ibid. 60: 582—584,

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Edited by A, K,