Plant Physiol. (1983) 72, 204-2110032-0889/83/72/0204/08/$00.50/0

Characteristics of Sulfate Transport Across Plasmalemma andTonoplast of Carrot Root Cells'

Received for publication July 26, 1982 and in revised form January 18, 1983

JOHN CRAMSchool of Biological Sciences (A 12), University of Sydney, New South Wales 2006, Australia

ABSTRACT

Compartmental analysis of "SO4-2 exchange kinetics is used to obtainS042- fluxes and compartment contents in carrot (Daucus carota L.)storage root cells, where 2 to 5% of the S042- taken up is reduced toorganic form. The necessary curve fit is verified by (a) consistencybetween 'content versus time' and 'rate versus time' plots of washout data;(b) agreement between loading and washout kinetics; and (c) correctidentification of the fastest exchange phase as being from extracellularspaces.

Sulfate is actively transported up an electrochemical potential gradientat both plasmalemma and tonoplast. The plasmalemma influx is from 2 to10 times higher than the tonoplast Influx, is much greater than the S042reduction rate, and would not lmit the rate of either. This is consistentwith the finding that the plasmie a influx is not regulated by internalS042- or cysteine (Cram 1982 Plant Sci Lett, in press).

Both SOt influxes rise with only limited saturation as the extenalS042- concentration increases up to 50 miHmolarity. Both effluxes appearto be passive, with extensive recyclng in the plasmalemma influx pump.S042- permeability is about 10-11 meter per second at both membranes.The high, nonlimiting fluxes of S042- at the plasmalemma relative to

the tonoplast (found also in Lemna; Thoiron, Thoiron, Demarty, Thellier1981 Biochin Biophys Acta 644: 24-35) contrasts with S04'- fluxes inbacteria and with Cl( fluxes in plant cells. Their implications for work oncharacteristics and regulation of S042' uptake in roots and tissue culturesare discussed.

Sulfate is the second most abundant of the elements which aretaken up as ions and metabolized (nitrogen being the most abun-dant) (8). Understanding the mechanism and control of its uptakein relation to long distance transport, storage and mobility (20,28), nutrient balance (11, 24), and metabolism (1, 13, 14, 24) inplants demands that fluxes of S042- and of organic S compoundsacross individual cell membranes and between metabolites shouldbe measured. In particular, in the vacuolated higher plant cell, thekinetics and regulation of s042- flows across the plasmalemmaand subsequently either to the vacuole or to reductive assimilationproducts can only be studied using such measurements.Compartmental analysis of the kinetics of 3S exchange is the

only available method for measuring and thus characterizing thesefluxes. Separation of vacuoles from protoplasts may also provideestimates of steady state distributions, but cannot help directlywith flux estimations because these demand instantaneous meas-urements of tracer distribution between vacuole and cytoplasm,while separating vacuoles from protoplasts takes a significant

' Supported by grants from the Australian Research Grants Schemeand the University of Sydney.

period of time.This paper provides an experimental justification of the use of

compartmental analysis of 'S exchange for estimating fluxes incarrot storage root cells. The method is then used to obtainthermodynamic and kinetic characteristics of S042- transport inthese cells, and the implications for the rate limitation and controlof So02- accumulation and metabolism are discussed.The first destination of s042- after uptake across the plasma-

lemma is reductive assimilation. In nonvacuolated bacteria, as invacuolated fungi and higher plants, it has been shown that reduc-tive assimilation is self-regulated by negative feedback from prod-ucts of s042- metabolism. Cysteine, for instance, feeds back to thefirst enzyme of s042- metabolism, ATP sulfurylase (which cata-lyzes the production of 'activated SO2-,' which in tUn is reducedin a sequence of reactions to S2- and then converted to cysteine,etc.) (1, 21). In bacteria, the study of mutants has shown, further,that this negative feedback also extends to the uptake of S042-across the plasmalemma, so that the uptake and reduction ofS042- are to some extent matched (1, 7, 21). Similar control ofs042- influx across the plasmalemma in higher plant cells has alsobeen claimed (15, 23, 24), although without supporting measure-ments of the plasmalemma influx.A priori, such a tight regulation of s042 influx across the

plasmalemma in vacuolated cells seems unlikely. If s042- uptakewere matched to S042 reduction in a vacuolated cell, then, forinstance, in the S-sufficient cell, s042- entry would be restricted;and yet 'luxury' S042- accumulation manifestly occurs in manyplants (11, 28). The present paper provides the first measurementsof S042- fluxes at the plasmalemma and the tonoplast of avacuolated cell over a range of external S042- concentrations, andshows that plasmalemma influx is so high that it would not limiteither reduction or accumulation of S042. Another paper (9)reports that the plasmalemma influx is not reduced by raising theinternal s042- or cysteine concentration, i.e. is not regulated byproducts of s042- metabolism. These observations agree with theconcept that a high, nonrate-limiting plasmalemma influx couldnot usefully be regulated, but that reductive assimilation andaccumulation in the vacuole are both independently self-regulated.

MATERIALS AND METHODS

Tissue of the storage root of carrot (Daucus carota L.) was cutinto squares (5 x 5 x 1 mm) or strips (50 x 5 x 1 mm) and washedin aerated distilled H20 with frequent changes for 4 or more d, tobring it to the state of rapid accumulation of ions before beingused in experiments. Samples of four strips were suspended fromstainless steel wire for tracer exchange measurements. They havethe advantage of minimizing handling and ullage problems, butthe disadvantage of being considerably more variable than theusual samples (2 to 2.5 g of randomly selected squares). Fluxes,internal concentrations, and electrical potential differences be-tween vacuole and external medium were measured by standardmethods (e.g. 4, 6), modified where necessary as described and

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CHARACTERISTICS OF SULFATE TRANSPORT

justified in this paper.Sulfate concentration was determined by measuring the optical

density of BaSO4 precipitated from an aliquot of the tissue extract.To 1 ml of the extract, suitably diluted, was added 3 ml of 200 mmBa acetate + 100 mm HNO3. With efficient shaking, a highlyreproducible standard was obtained. The use of agar (26) wasfound to be unnecessary and undesirable as all brands tested hadsignificantSO442- contamination.

Organic sulfur was separated from so42- after the latter hadbeen precipitated as BaSO4 and filtered off. A correction was

made to counts in precipitated 35so42- to allow for self-absorptionand differences in sample geometry.

RESULTS

Justification of Compartmental Analysis of 36S Exchange Ki-netics. A necessary condition for a compartmental interpretationof tracer exchange kinetics is that the tissue is at or very near asteady state (4, 29, 31). In particular, any rapid adjustment of thecytoplasmic phase after transfer from one solution to another musthave been completed. Preliminary experiments showed that cy-toplasmic 3SO42 exchanged with t1/22 of around 16 min, andtherefore, in all measurements, the tissue was pretreated in a

solution of the same chemical composition but not labeled with35SO42- for 100 min or more (>5 x cytoplasmic 11/2), by whichtime the cytoplasmic s042- would have reached a quasi-steadystate (or at least moved 97% of the way towards it). In this state,its concentration would be changing no faster than the slowlychanging vacuolar so42- concentration.To measure the kinetics of 3S exchange, the tissue was pre-

2 Abbreviations: t1/2, half-time for turnover of molecules in a compart-

ment; p.d., potential difference.

10

8

61E0.u

0

z

z0u

U)

I-0

4

2

100

treated in inactive solution (as above), then placed in a solution ofthe same chemical composition but containing 35so42- to labelcellular s042- and organic S pools, and finally transferred tosuccessive aliquots of unlabeled solution of the same composition.All 3S flowing to the external solution would be as s042- (27).An example of the time course of loss of 3S to the externalsolution is shown in Figure 1. Standard 'curve peeling' (4, 29, 31)revealed three exponential components, the slowest appearing inFigure 1, and the two faster components in Figure 2. Eachcomponent is characterized by the intercept at t = 0 (I), sometimesmisleadingly called the 'apparent content,' and the exponentialrate constant (k). Anticipating subsequent results, they will becalled vacuolar (v), cytoplasmic (c), and extracellular (f, fast)components of exchange.

It will be noticed that over 80%1o of the 35S activity is in the fasterexchanging components, and they are virtually unaffected by anyuncertainty in fitting a single exponential to the vacuolar compo-nent for extrapolation to short washout times. This is fortunate,since the slowest component of exchange was frequently morecurved than a single exponential would be. The curvature in theslowest component of exchange can be represented by the changein its rate constant (perhaps therefore better called its 'rate coef-ficient' in this case) over successive time intervals. The rateconstant kU (= rate of loss/average content) fell from a normalized100%o after 2 to 2.5-h washout to 57 ± 17% (SD, 45) at 3 h, and to42 ± 15% (SD, 37) at 4 h. The average value over the period 2 to4 h was extrapolated to t = 0 for subtraction from the total in thetissue to obtain the faster components of exchange.The higher values of ks at the beginning of the slow phase of

washing out are probably due to a contribution of organic Sexchanging more slowly than the bulk cytoplasm and faster thanthe vacuole. If this is the case, then the correct value of ks fordescribing vacuolar turnover is the final, lowest value, which by

200 300

TIME/MIN

FIG. 1. The time course of loss of 3SO442- from 'SO42--labeled carrot root tissue. Five-d water-washed tissue was pretreated for 100 min in I mMNa2SO4 + 0.1 mm CaSO4, labeled for 60 min with 35S042 in a solution of the same chemical composition, then allowed to exchange 3SO442- with

successive aliquots of the same unlabeled solution. The final 35S content of the tissue was measured, and the 35S content at earlier washout times was

calculated and plotted on a log scale against washout time. The final slow phase was extrapolated to zero washout time, and the extrapolated content

was subtracted from the total. The difference at each point is plotted in Figure 2.

TOTAL IN TISSUE

0

_

0~~0~~~ ~ ~

.

I a I

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Plant Physiol. Vol. 72, 1983

E

0.

I--

z

~~~~~~~~~~~~~510z

O TIME/MIN

n 1.

0*5

20 40 60

TIME/MIN

FIG. 2. Tissue content of3S after the slowest component of exchangehas been subtracted as in Figure 1, plotted on a log scale against washouttime. The intermediate phase was extrapolated and subtracted from theremaining total, and the difference is plotted on a log scale against washouttime in the inset. The slopes give tl/2 for exchange of the two componentsof 16 and 2.6 min.

Table I. Consistency in Curve Fitting to3S Washout DataRandomly selected sets of exchange data were plotted as content versus

time and as rate of loss versus time, and rate constants and intercepts at t= 0 of the two fastest exchange components of the two types of plot were

measured and their ratios were calculated. A ratio of unity indicatesinternal consistency of a fitted parameter. Values are quoted as mean +SD (number of replicates in parentheses).

Cytoplasmic Initial FastComponent Component

kcontent/krate 0.94 ± 0.12 (8) 1.06 ± 0.18 (7)Icontent/(Iratelkrate) 0.97 ± 0.07 (8) 1.06 ± 0.13 (7)

extrapolation is estimated to be 20 to 30%o of the value at 2 to 2.5h of washing out. This extrapolated value was the one used in fluxcalculations.Checks on the Validity of the Curve Fitting. (a) If the plot of

'log (total 35S content) versus time' (Fig. 1) is correctly fitted bythe sum of three exponential terms, as described in the previoussection, then the plot of 'rate of loss of3S versus time' should alsobe fitted by three components with the same rate constants butwith intercepts kU I,, k,.hI,, and kf.If, respectively. To test this,eight sets of exchange data from four sets of conditions werepicked at random, and values of these parameters were obtainedfrom the two types of plot.

Table I shows that for the two faster components the exponentialfunction is consistent in its fit to the two types of plot, thuseliminating the possibility of erroneous fitting due to the experi-mental scatter in the data points (cf. Ref. 10 and MacRobbie's

improved method [18]).(b) The second check on the kinetics is to see ifinflux and efflux

time courses agree. If they do, then after longer labeling periodsin 35SO42- the sizes of the intercepts should rise with a [1 - exp(-kt)] time course, k being equal to that obtained from effluxkinetics. Figures 4 and 5 show that the vacuolar and cytoplasmiccomponents agree well with this expectation. The extracellularcomponent (Fig. 3) is intrinsically more variable, but there is nosystematic deviation of If from expectation.

(c) The fastest component of 3mQSO42- exchange in living tissueis made up of an initial very fast phase, which must be fromunstirred surface layers and is too variable for profitable analysis.This is followed by the fastest exponential component which mustcorrespond to diffusion from extracellular spaces within the tissue.If this is the case, then in dead tissue 3SO42- should exchange asa single exponential with approximately the same t1/2 but withcontent now equal to 100% of the water-filled volume of the deadtissue, and neither of the slower exchanging components shouldbe present. The extracellular component in live tissue should alsohave content proportional to the external concentration, and equalto that measured with C1- (4), and a rate constant which isindependent of external concentration and twice that for C1-(since the self-diffusion coefficients of so42- and C1- are in theratio 1:2; Ref. 2). These expectations were all confirmed (TableII).There are two important corollaries of the third point. (a) Since

the fastest component of exchange represents the -extracellularS042-, the two slower components must ipsofacto represent all ofthe cellular S. (b) The correct fitting of the fastest exchangecomponent depends on first fitting the slower components cor-rectly. Inasmuch as the fit to the fastest component is evidentlycorrect, the fit to the cytoplasmic and vacuolar components mustalso be correct.

Exchange of ssSO42- with Organic S. After 1 to 3 h loading in35so42-, 2.5 to 6% of the 'S in the tissue was in water-solubleorganic S compounds, with little in insoluble S. There was nosignificant difference between aseptic and ordinarily preparedcarrot tissue, showing that bacterial reduction of SO02- is insignif-icant. Anticipating the results ofTable III, the cytoplasmic specificactivity (se) during loading is ;0.8 times that in the externalsolution, giving the flux of S042 into organic S of 2.1 ± 1.1 nmolg-1 h-1 (mean of five sets of experiments, ±SD) in 0.5 or 1 mMexternal S042.The back flux of 35S from organic S to s04-2- probably occurs

through cysteine (e.g. 14). When the tissue was in 1 mM L-cysteine,5(steine influx to the cytoplasm was 137 nmol g-1 h-1, the flux ofS from cysteine to s042 was 9 nmol g-1 h-' (and the net flux of

S from cysteine to S042- was 6 nmol g-1 h-'), and the cysteinecontent of the cytoplasm was 90 nmol g-1. (These results wereobtained by a similar compartmental analysis of tracer exchange,but the results are not presented here in full.) In the absence ofexternal cysteine, the cytoplasmic cysteine concentration will bevery nearly zero (12). If it is proportional to the rate of appearanceof cysteine in the cytoplasm (via influx from the external solutionor reduction of SO42), then in 1 mim K2SO4, with the rate ofproduction of cysteine being about 2 nmol g-1 h-1, the cytoplasmiccysteine concentration would be just over 1 nmol g-1 and the flowof S from cysteine to S042- would be of the order of 0.1 nmolh-'. This rate is insignificant compared with the total rate of Sloss from the tissue, which at the end of the washing out period isaround 5 nmol g9- h-1 x (s[0.15J) = 0.75 nmol g-1 h , and isalso insignificant compared with the flow of "S into cysteine.

This suggests that the cytoplasmic phase, which must be theorigin of the curvature in the slowest phase of washing out, mayin fact be not organic S but rather exchange of S042- in anorganelle.The flow of 35S into organic S in carrot root cells is therefore

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CHARACTERISTICS OF SULFATE TRANSPORT

Table II. Extracellular 35SO42- and Cl- ExchangeComparison of the fastest component of 35SO42- exchange in living

carrot tissue with that of 'SO42- in dead tissue and with that of 36Cl- inliving tissue. Values ofexchange parameters were obtained as described inFigures 1 and 2. Values are quoted as mean i SE (number of replicates inparentheses).

Exchange t1/2 Content

nun % tissue volFastest 35SO42- in living tissue 2.3 ± 0.1 (41) 4.0 + 0.4 (31)Total 3SO42- in dead tissue 2.6 ± 0.3 (3) 99 ± 6 (3)Fastest 36Cl- in living tissue 1.2 ± 0.1 (18) 6 t 1 (13)

essentially irreversible, and can for calculation be lumped with.* . . . . inflow to the vacuole. Whether this is also true in photosynthetic

10 20 30 40 50 60 tissues reducing significant quantities of S042- (e.g. Lemna; Ref.TIME/MIN 27) remains to be determined. Comparable rates of conversion of

cysteine to s042- have been found in roots of the legume Macrop-The intercept of the fastest component of exchange (the com- tilium atropupreum (Clarkson, Smith, and Vanden Berg, personal

hown in the inset to Fig. 2) plotted against loading time from 0 to communication), and rather higher rates have been found in

T'he dashed line shows a [1 - exp(-kt)] rise with k equivalent to tobacco tissue culture (25).

.6 min. This demonstrates agreement between the kinetics of Estimation of S04 Fluxes and Compartment Contents within

(Fig. 3) and the kinetics of washing out (Fig. 2). the Cell. It will be assumed that 'cytoplasmic' and 'vacuolar

exchange components correspond to turnover in these compart-ments, that the cytoplasm exchanges as a whole because of veryrapid turnover in organelles due to their high surface to volumeratio, and that cytoplasm and vacuole are arranged in series. The

"CYTOPLASMIC INTERCEPT flux of 'S from cytoplasmic SO02- to organic S will be lumpedwith the flux of SO4 from cytoplasm to vacuole inasmuch as

_- l both fluxes are from the same pool. Then, having established aquasi-steady state by pretreatment, Equations I to 9 hold (see e.g.4, 29, 31 for derivations) with the foliowing definitions.

Measured parameters comprise: k (h), rate constant of an

oo exchange component; I (nmol g-'), intercept of an exchangecomponent at the beinning of washing out; ti,, (h), influx time in

35SO423 ; QT (nmol g ), totalS242- in the tissue. Estimated valuescompnse: 40u (etc.) (nmol g-1 h-1), flux from cytoplasm to vacuole

X (etc.); Qc, Q, (nmol g-1), chemical content of cytoplasm andvacuole; s,, s,,, specific activity of cytoplasmic or vacuolar S042

0 20 30 40 50 60 relative to the external solution.TIME/MIN Subscripts v, c, andfrefer respectively to vacuolar, cytoplasmic,

I. Treatments were as in Figure 3, but the cytoplasmic intercept and extracellular (fast) components ofexchange or compartments,I and the 11/2 for labeling is 15 min. The kinetics of labeling (Fig. and subscript o refers to the external solution.f washing out (Fig. 2) agree. The quasi-steady influx of tracer to the vacuole is given by

40

30

I,

"'N

20

10

10 20 30 40 50 60

TIME/MIN

FIG. 5. Treatments were as for Figures 3 and 4, but the vacuolarintercept is plotted and the 11/2 for labeling is 7 h. The kinetics of labeling(Fig. 5) and of washing out (Fig. 1) agree.

(1)OWv = Iv/tin5 oOC-

CU (= OCV-SC)'0CO + sCV

The product k,Ic gives

kc-Ic-= *oc4coloo + 'Wv

(2)

(3)

The plasmalemma influx is obtained by adding Equations 2and 3

4oc = Iv/tm + kcIc (4)The ratio of tonoplast influx to plasmalemma efflux is obtainedfrom Equations 2 and 4

4,cv = Xv = lu/ti

40co ooc- ov kc-IcThe plasmalemma efflux is given by

=Ocz kc-Ic + ku-Qv*oc4*co + oVC- 0CO= o + oc+kCO +'c*C +'+ CU

(5)

(6a)

(6b)

EC

501

EXTRACELLULAR CONTENT 0

0 ~~~~~~~01 0

// 0

I

FIG. 3ponent sl60 min. It1/2 of 2.

labeling (

50I0Ec

25

FIG. 4is plotted4) and of

"VACUOLAR" INTERCEPT

0-V

I s , , .0 . I-V.

-V

OA-,,0.11~-I 10#1~~~~~~~-

*,-0.11~ ~ -

..or.00*~~~-a -IV

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Plant Physiol. Vol. 72, 1983

As already noted, ku is difficult to measure with certainty inthese experiments (although this uncertainty is not sufficient toaffect the estimation of ke and I).

Alternative to Equations 6a and 6b, the plasmalemma effluxcan be obtained from the net influx and oc

4co = Soc - (net (7)Again, 4et is difficult to measure with accuracy because it is so

small. Nonetheless, ,t has been measured in a few conditions,and it is always positive and approximately equal to 4Ov. Hence,limits can be placed on the value of 0. (and consequently on OC4).

Equation 6a gives the minimum value of 4,, as kIc LI (and thesame result is obtained from Equation 7 if Inet = 4ov). Equation 7gives the maximum value of 4co (when net = 0) as equal to fo.Since, when it has been measured, 4et is approximately equal to00, we shall generally take O. to equal oc- o, except where areliable value of Ot or of k, was obtained in a particular experi-ment. The tonpolast influx is then obtained from Equation 5. Thetonoplast efflux is obtained from the quasi-steady state conditionof no net flux into the cytoplasm

'O'C + O'C = Oco + Oc, (8)The cytoplasmic content is obtained from the relationship

QC = IACV (9)

and the vacuolar content is approximated by the total S042- inthe tissue (QT), since QC is small relative to QT.Thermodynamic Characterization of SO4' Fluxes. Values of

s042- fluxes at the plasmalemma and the tonoplast and of cyto-plasmic and vacuolar s042- contents in 1 mM s042- solutionswere measured as controls in a number of experiments. Thevariability between different batches of tissue was considerable.(SO42- fluxes are in this respect unlike C1- fluxes which vary littlefrom batch to batch.) In addition, the distribution of mean valuesin different batches of tissue followed a skewed, not a Gaussian,distribution, with a few high values of Ic and kv particularlybiasing the distributions. However, variation in seven batches oftissue was more or less normally distributed, and the average andstandard deviation of the means of these are presented in TableIII, together with the net influx and electrical potential differencebetween the vacuole and the external solution measured sepa-rately.Two other batches of tissue were significantly different, having

6- to 7-fold higher values of Ic (54 and 60 nmol g-1 comparedwith the average of 8.6 nmol g-' for the others), and a 4-foldhiqher value of kv (0.042 h-' compared with an average of 0.010h). These corresponded to 5-fold higher plasmalemma fluxesthan the average of the other batches, but to tonoplast fluxes ofcomparable size. Results for these batches are shown in Figures 1to 5, and in Table IV.

Table III. Average Values ofMeasured Parameters of 35SO42- Exchangein Excised Washed Carrot Storage Tissue in I mM Na2SO4

The average of the means of seven experiments are listed, together withSE. QT, 4,,, and Eo were measured in separate experiments.

Parameter Measured Value

I/tA 20 + 4 nmol g-' h-'ke 2.6 ± 0.2 h-'i., 8.6±2.1 nmolg'kv, 0.01 + 0.008 h-QT 548 + 62 nmol g-'h'I0-t 15 ± 5 nmol g-'h'Evo -l130 mv

In the other seven batches of tissue, fluxes do not all varyindependently. The plasmalemma fluxes change together, as dothe tonoplast fluxes, but plasmalemma and tonoplast fluxes werenot correlated.The calculated mean fluxes for these seven batches of tissue are

given in Figure 6. The standard error of the mean of the plasma-lemma fluxes was 12 to 16%, that of the tonoplast fluxes was 30 to45%, and that of the cytoplasmic content was 22%. These meanvalues will be used for the purposes of thermodynamic analysis.The same conclusions would be reached by examining the valuesof the other two batches of tissue.The cytoplasm occupies about 3% and the vacuole about 80%

of the tissue volume, so the average s042- concentration will beabout 0.7 mm in the cytoplasm and 0.6 mm in the vacuole. Theelectrical p.d. will be assumed to occur almost entirely across theplasmalemma. From these values, it can be calculated that S042-ions move up an electrochemical p.d. of 24 kJ mol-' on enteringthe cell, and must therefore be actively transported inwards acrossthe plasmalemma. The situation at the tonoplast is clearer inS04- loaded tissue. Here, there is a net SO2 influx when thecalculated cytoplasmic and vacuolar concentrations of s042- are0.2 and 13 mm, respectively. The vacuolar electrical p.d. is slightlymore negative than in the nonloaded tissue, and it is againassumed that this is mainly located across the plasmalemma. Fromthese values, it can be calculated that s042- moves in across thetonoplast up an electrochemical p.d. of 10 kJ mol-', and must beactively transported inwards here also.

Kinetic Characterization of SO4'- Fluxes. Fluxes of s042 incarrot tissue from 0.1 to 50 mM Na2SO4 are shown in Table IV.The main feature of these results is that the plasmalemma fluxes

A. CARROT, SO0

A CYT , VAC

I FM 0\-7mM o-5mM

0ONV

B. LEMNA, S04-CYT VAC

63 C- 2

034-3m 1

0-5mMW 0-3mM 1-3mM

C. CARROT, Cl

FIG. 6. Comparison of S042- fluxes in carrot with S042- fluxes in Lminor and with C1- fluxes in carrot. Values taken from Table I (A),Thoiron et al. (Ref. 2) (B), and values for C1- in carrot under the sameconditions (C) are calculated from original data. Note the similaritybetween the S042- fluxes in carrot and Lemna, and the difference fromC1- fluxes in carrot. All fluxes are quoted as nmol g-' h-'.

Table IV. S042- F7uxes over a Range ofExternal S042- ConcentrationsCarrot tissue was pretreated in nonlabeled solutions of the appropriate

concentration for 75 min, then placed in the same solutions labeled with'SO42- for 67 min before measuring tracer efflux. Fluxes, etc. werecalculated from exchange data as described in the text. A value of 0.005h-' for kv was assumed, but uncertainty in the value ofku has no qualitativeconsequences for the conclusions drawn. Values quoted are means ofduplicate calculated values which differed by 30% or less. The fastestcomponent of exchange had a content which was proportional to theextemal concentration, was equivalent to 6% of the tissue volume, andhad a tl/2 of 2.5 min independent of external concentration.External Na2SO4 0 0l)c Om Ql C.Concentration

mM nmolg ' h' nmolg' mM

0.1 11 10 1 0 3.9 0.131 158 141 23 6 66 2.2

10 1,580 1,447 142 9 662 2250 11,566 11,143 436 14 3,000 100

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CHARACTERISTICS OF SULFATE TRANSPORT

are 1 order of magnitude higher than tonoplast fluxes at allexternal concentrations, and that the cytoplasmic s042- concen-tration is about twice that in the external solution over the wholerange, increasing more or less in proportion to the external solutionconcentration. Other batches of tissue were qualitatively similarin having the plasmalemma influx greater than the tonoplastinflux, although generally the plasmalemma influx was twicerather than 10 times the tonoplast influx at 1 mm external s042-(Fig. 6).Both the plasmalemma influx and the tonoplast influx show

surprisingly limited saturation at high external s042- concentra-tions. Use of Equation 10 (below) shows that even at 50 mMexternal s042- concentration, the passive s042 influx is negligi-ble. Overall influx therefore appears not to be composed of asystem saturating at low external s042- concentrations plus apassive influx increasing at higher external s042- concentrations.To examine the possibility that plasmalemma s042- efflux is

passive, the permeability coefficient (PS042-) can be calculatedfrom the results in Table IV together with measured values of theelectrical p.d. at each external So42- concentration taken byextrapolation from Reference 6. The equation used (e.g. Hopeand Walker, Ref. 16) is

zFE,. [SO42-]c

RT (epzFE.'](0ex \RT )

Values of Pso42- are calculated to be 1.0, 1.1, 1.9, and 5.6 x 10-1m s_' at 0.1, 1, 10, and 50 mm external so42-, respectively. Asimple assumption would be that PS042- is constant at 1 x 10"1 ms-5 at all external concentrations, but that a significant fraction ofthe efflux at higher external S042- concentrations results fromrecycling in the pump. At 50 mm external S042-, 82% of the efflux(or 9 ,umol g-1 h-') would on this interpretation be recycling inthe pump.The tonoplast efflux is the most uncertain of the estimates

because of the difficulty in measuring accurately either the netinflux or the final quasi-steady tracer efflux, one of which isnecessary for the estimation of 4,,,. Acknowledging this uncer-tainty, and assuming that the tonoplast electrical p.d. is around 5mv, then Pso 2- for the tonoplast is calculated to be of the order of1 to 2 x 10-t' m s-1, which is close to the value for the plasma-lemma.

Interaction of SO42- Fluxes with Fluxes of Other Ions. s042-influx was measured in 1 mM CaSO4, 1 mm Na2SO4, 1 mm K2SO4,and 1 mm Na2SO4 with added 8 mLm Na+ or K+ benzenesulfonate.There was no significant difference between the plasmalemmainfluxes or between the tonoplast influxes in any ofthese solutions.Since influx is not saturated at 1 mm external s042-, these changesof solution would have shown up any limitation on So0 influxdue to the obligatory link to the concomitant influx of a particularcation.

Similar negative results were obtained when KCI or KNO3 wasadded to an external solution of 1 mM s042-. There was nosignificant effect on s042- influx across the plasmalemma or thetonoplast. The possible linkage to H+ influx has not yet beeninvestigated.

After excision, So4 influx increases over 4 d, as does C1-influx. The increase in S042- influx between 0 and 4 d varied from2 to 13 times. This variation makes it impossible to compare thechanges in so42- influx with the changes in C1- influx followingexcision from the storage root.

DISCUSSION

The kinetics of exchange of S042- (or other metabolites) in aplant cell are intrinsically more complex than those ofC1- or othernonmetabolites because of the presence of chemical as well as

physical compartments. In carrot root tissue, however, the backflux of S from organic S to s04.2- was shown to be negligible, andconsequently, S only flows one way between So42- and organic S.In kinetic terms, the addition of an irreversible flux from So2 toorganic S does not add any compartments to the model used forS042- fluxes in carrot.However, in general, the addition of one or more metabolic

compartments exchanging 3S with So42- would make the modeltoo complex to analyze from exchange of tracer in the system asa whole (ie. from influx and efflux kinetics alone). But at thesame time as it increases the complexity ofthe kinetics, a metaboliccompartment provides extra access to the system since it can besampled independently. Thus, future studies of the metabolismand transport of sulfur will combine measurements ofthe changesof 3S in organic compounds (as made by Giovanelli et aL, 13)with measurements of influx and efflux kinetics as in the presentpaper and in that ofThoiron et aL (27). From these measurements,chemical as well as physical flows of S will be calculable.An example of the interrelationship of chemical and physical

flows is the initial lag in the build up of wS in cysteine whichwould be related to the rate of turnover of 35SO42 in the cyto-plasm. In Chlorella, the observed lag is equivalent to a cytoplasmicturnover t1/2 of 4 s (calculated from Fig. 6 of Giovanelli et al.[13]). Preliminary attempts with carrot root tissue to see whetherthe lag in the build up of 3S in organic S corresponded to thatpredicted from the value of kc, obtained from washout measure-ments failed because of the variability of the tissue.

It seems appropriate to recall once again the necessity for doingsuch experiments at or very near a steady state (4, 29, 31), as hasbeen emphasized in recent work on So0 and sulfur transportand metabolism (13, 27). If the system is not at a steady state,then there is no a priori basis for interpreting exchange kinetics.An exchange curve at a non-steady state can only too easily befitted by the sum of two exponential components. However, thereis no method of telling if these are describing the exchange oftracer in a two compartment system or whether they fortuitouslycoincide with the change in a single chemical flux with time orwith the exchange in a more complex system.

Characterstics of SO42- Fluxes in Carrot Root Tissue. Thecharacteristics of So02- fluxes in carrot root cells can be summa-rized as follows. The plasmalemma influx is predominantly active,but surprisingly shows little sign of 'saturation' at higher externals042- concentrations. It is apparently not subject to feedbackcontrol from either internal S02- or cysteine (9). Plasmalemmaefflux can be described as a passive flow at low external S042-concentrations (Pso42-= 10"1 m s-'), with recycling in the influxpump becoming significant at higher external s042 concentra-tions.The tonoplast influx also appears to be active, and shows signs

of saturation at high external and cytoplasmic s04.2- concentra-tions. It is subject to feedback control from vacuolar S042- (9).The tonoplast efflux, which is difficult to measure accurately, canbe accounted for as a passive flow with the tonoplast permeabilitycoefficient being about the same as that of the plasmalemma.The main physiological characteristics of so42 in carrot root

cells are: (a) The plasmalemma fluxes are 2 to 10 times higherthan the tonoplast influx, so that the plasmalemma influx doesnot rate-limit entry to the vacuole. The same applies to Sot2-reduction. More So-.2- flows into the cytoplasm than flows intothe vacuole or is reduced, and the excess flows out again acrossthe plasmalemma.

(b) The Sot2- concentration in the cytoplasm rises concomi-tantly with that in the external solution, rather than being heldfairly constant like C1- (4). This can be understood from the flux-concentration relationships in Table IV. Cytoplasmic SOt2- ac-cumulation (which will reach a steady state about as fast as tracerequilibrates, viz. 90 min) is therefore best viewed as a modified

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2Plant Physiol. Vol. 72, 1983

pump and leak system.(c) Accumulation of S042 in the vacuole, by contrast, may well

include an element of homeostasis (9). It is determined mainly bythe tonoplast fluxes, not the plasmalemma fluxes. As the concen-tration of so42- in the vacuole increases, the active tonoplastinflux will decrease (9), and the passive tonoplast efflux willincrease. When they are equal there will be no further change.Increasing the external so42- concentration, and consequentlys042- influx, would tend to cause the vacuolar s042- concentra-tion to increase, but the negative feedback from vacuolar s042- tothe tonoplast influx (9) may override this tendency, as is the casewith homeostatic C1- accumulation (W. J. Cram, in preparation).It is difficult to test this because the very low s042- fluxes resultin the steady state being reached only after a very long time.

(d) When external S042- concentration is over 0.5 mm, thecytoplasmic s042- concentration will be above 1 mm, which iswell above the Km for ATP sulfurylase (1). Hence, So42- supplywill not limit s042- reduction when the external s042- concentra-tion is above 0.5 mm. This is the basis of point (a) above.

Contrast with Tightly Regulated S042 Influx in Microorgan-isms. Only a 'rate-limiting' step in a sequence can usefully becontrolled. In bacteria and fungi, the rate-limiting step in s042-utilization is probably the influx across the plasmalemma, and itis frequently found that the influx is regulated by negative feed-back from end product(s) of S042- reduction (1, 21), resulting ina more or less strict stoichiometry between uptake and reduction,thus matching the supply ofS042 to the demand for its reduction.By contrast, in the vacuolated higher plant cell (represented by

carrot and Lemna), the plasmalemma influx appears not to be ratelimiting for reduction (or accumulation), and neither is it regulatedby internal s042- or cysteine in carrot (9). This is as expectedinasmuch as plasmalemma influx supplies s042- both for vacuolaraccumulation and for reduction. The parallel has already beendrawn between self-regulation of s042 accumulation and ofreduction in a vacuolated cell, and self-regulation in a branchedbiochemical pathway (9).

Similarty to SO22- Fluxes in Lemna Minor. Sulfate fluxes atplasmalemma and tonoplast have been measured in L minorgrowing in a culture solution containing 0.54 mm s042- (27). Thevalues are shown in Figure 6 alongside those for carrot root.Lemna differs from carrot in reducing about half the s042- ittakes up, whereas carrot reduces only 2 to 5%. Otherwise, thes042- fluxes in the two tissues are remarkably similar at a com-parable external s042- concentration. In particular, the plasma-lemma influx is bigger than the fluxes inside the cell, and in bothtissues, about 60% of the s042- influx returns to the externalsolution across the plasmalemma. The plasmalemma influx canhardly limit the rate of s042- reduction or accumulation in thephotosynthetic, S042--reducing, Lemna cell, just as the plasma-lemma influx cannot be a limitation in the nonphotosynthetic,slowly SO42--reducing carrot root cell.

Contrast with Cl Fluxes in Carrot. The high values of plas-malemma s042- fluxes compared with those for the tonoplast incarrot root cells can be further emphasized by comparing themwith C1- fluxes in carrot root tissue (Fig. 6). Sulfate fluxes are

lower than C1- fluxes, but there is also a marked qualitativedifference in the proportion of the ions entering the cell whichflow to the vacuole. In the case of So42-, over half returns to theexternal solution, whereas with C1- the majority flows straight oninto the vacuole. The latter condition is also true of C1- and otherions pumped inwards across the plasmalemma in many other cells(16, 19). The ratio of tonoplast influx to plasmalemma efflux is inthe region of 30 times lower with S042- than with Cl- in carrotroot tissue.

Cr- taken up by carrot and other cells is almost exclusivelydestined for accumulation in the vacuole. S042- taken up by carrotcells is destined partly for accumulation in the vacuole, partly for

reduction, but the majority is destined for recirculation to theextracellular spaces.

hplcatlns Of H Plsl S042- FluxeS for KineticStudies of SO42- Influx. The most frequent method of measurngS04-2 influx in studies of its kinetics consists of placing the tissuein 3S042- solution for 10 to 60 min followed by washing for 10 to30 min (15, 17, 22, 30). This provides a measure of the quasi-steady influx to the vacuole (5, 27), but does not, as has frequentlybeen assumed, necessarily provide a measure of the plasmalemmainflux. Because of the high plasmalemma s042- fluxes, the quasi-steady influx will approximately equal the tonoplast influx incarrot and Lemna. Kinetic studies using the quasi-steady S042-influx may therefore need re-interpretation if other cells aresimilar.A particularly important case is that of the regulation of s042-

influx in tissue cultures of Nicotiana. In this tissue, the quasi-steady influx of S042- was reduced in high S cells, and this wasinterpreted as feedback inhibition ofplasmalemma influx (15, 23,24). This would imply that the plasmalemma influx is rate limitingfor So2- accumulation in the vacuole and also for the rate ofS042- reduction, which would contradict the conclusions fromcarrot and Lemna. However, if in Nicotiana the plasmalemmas042- fluxes are as high as in Lemna and carrot, then under theconditions used in the Nicotiana experiments, the uptake ratemeasured would have been equal not to the plasmalemma influx,but rather to the tonoplast influx plus the rate of reduction. Thefeedback effects observed would, furthermore, have been fromvacuolar s042- to tonoplast influx plus that from S metabolites toS042- reduction (which would also account for the somewhatloose correlation between 'influx' and cellular so42 concentra-tion). The three sets of data are therefore probably compatiblewhen the rate-limiting steps involved are taken into account.Other Characteristics and Implications of the High Plasma-

lemma SO4I-Ifux in Carrot Root Cels. s042 influx in carrotdiffers from that in some algae (19) in having no specific cationdependency. It also shows no competition with NO3, as previ-ously reported in other higher plants (e.g. 17).Development of S024 influx with time after excision of carrot

root tissue places the process alongside many others which developsimilarly, but does not by itself illuminate the physiology ofSO4 -transport and reduction in the cell.Cl- ions differ from s042- ions in appearing to be kept at a low

concentration in the cytoplasm even when the external Cl- con-centration is high (4). With SO42-, by contrast, the high plasma-lemma fluxes mean that the cytoplasmic s042- concentration isonly low if the extracellular SO4 - concentration is low. Sulfateaccumulation in extracellular spaces in the shoot might thereforelead to So42- toxicity rather more easily than would be the casewith C1-. The function of vacuoles in accumulating S042- maytherefore primarily be related to clearing the extracellular spacesof s042- (detoxification), rather than to serving as a storagereservoir (a concept which is difficult to accept on other groundsalso [9]).

Finally, the high plasmalemma influx of S024 in parenchymalcells may have a parallel in a high plasmalemma S042- influx tosieve tubes. This is supported by the observation ofhigher influxesof S042- to phloem tissue than to adjoining parenchymal cells (3).In sieve tubes, the tonoplast is absent, but in parenchyma cells, itis present and would limit the influx that was measured.

Acknowledgments-I am gateful to Pamela Hodson and to Dale Russ for technicalassistance.

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