Physiol. Plant. 50: 199-207. 1980 EFFLUX OF K+(«<'Rb) AND FROM ROOTS 199

Effects of ionic strength and relative humidity on the efflux of K" ( ^Rb) and^ ^ ) from roots of intact seedlings of cucumber, oat and wheat

By

PAUL JENSEN and ANDERS KYLIN

Department of Plant Physiology, University of Lund, Box 7007, S-220 07 Lund, Sweden

(Received 18 January, 1980; revised 10 June, 1980; in final state 19 June, 1980)

AbstractSpring wheat (Triticum aestivum L. cv. Svenno), oat (Avenasativa L. cv. Brighton) and glasshouse cucumber (Cucumissativus L. cv. Bestseller Fl) were cultured for a week after ger-mination on complete nutrient solutions of three different dilu-tions (1, 25 and 50% of the full strength medium). K-'(*''Rb) and"'Ca were present during the whole culture period. Relativehumidity (RH) was 50% except during the last day, when halfthe material was transferred to 90% RH. Efflux of labelled ionswas then followed during eight hours on unlabelled solutions ofthe same composition as before, and at both 50% and 90% RHin the atmosphere. - Uptake of K" (*''Rb) during growth tendedto be saturated in the 25% medium. Contrariwise, the level ofCa " in the roots increased continuously with strength of themedium. At low concentrations cucumber roots were higher inCa " than roots of oat or wheat, whereas all three species showedsimilar levels of Ca " in 50% medium. - At the lowest ionicstrength, smooth efflux curves were obtained that could be resol-ved according to the three-compartment theory. At higher ionicstrength, irregularities were observed, and more for Ca' '' thanfor K*; but for practical purposes compartment analysis with thesame time constants could be applied as for the lowest concent-ration. - Discrimination between K" and Rb" differed betweenthe roots, but not much between the shoots of different species.The roots of oat and wheat took up Rb" preferentially over K " inthe 25% and 50% media; whereas K"*" was preferred over Rb ' orlittle discrimination made in 1 % medium and for cucumber. Theshoots generally showed less discrimination than the roots. Themain variability in discrimination between K" and Rb" thus ap-pears to be localized in the tonoplasts of the roots cells. - LowRH around the shoots increased efflux of K' (**'Rb) from thecytoplasm and vacuoles of the root cells as compared to theefflux at high RH. DNP (2,4-dinitrophenol) in the medium hadthe same effect as high RH around the shoots. The signal systemthat must exist between shoots and roots is discussed as a re-sponse to "drought" conditions. In relation to investigations ofothers, it is assumed that the effect of DNP may indicate that partof the chain between roots and shoots consists of metabolicallyinfluenced sites, whose output is influenced by the rate of watertransport.

Introduction

A parallel behaviour has been shown between ecologicaland agricultural properties related to the soil and ATPaseactivities of the microsomal fraction of root homogenates(Kahr et al. 1977, Kawasaki et al, 1979). Physiologicalmeasurements appear necessary in order to understandproperly the significance of the parallelisms mentioned.As a first step in this direction, we have started a series ofmeasurements of ion efflux from intact plants.

At an early stage of the work, it was found that theefflux curves showed irregularities that are not accountedfor by the common three-compartment theory in the for-mulation of Pitman (1963). In a way this is not surprisingsince the whole-plant system will evidently contain com-plications both because of the histological structures andbecause of flow of water through the root in the directionoutside-in. To get perspectives on these complications,three levels of ionic strength and two different levels ofrelative humidity in the atmosphere have been used.Some experiments on the effect of 2,4-DNP have alsobeen done.

It is the scope of the present paper to summarize theobservational data on efflux of K' (*''Rb) and Ca "*" fromcucumber, spring wheat and oat, which have all beentouched upon in the investigations cited above. The exis-tence of a physiological sequence starting from relative airhumidity and ending with regulation of efflux ofK^(**'Rb) from the cytoplasm and vacuoles of root cellsseems unequivocally demonstrated. The implications ofthe findings will be discussed in relation to the data ofothers.

Key-words: Potassium; calcium; efflux: compartments; roots;ionic strength; relative humidity; 2,4-dinitrophenol;isotopic discrimination; cucumber; wheat; oat.

Abbreviations: [K" (**Rb)],oot, K' (**Rb) concentration in theroot; [Ca^*(''*Ca)],oo,, Ca ''C*'Ca) concentration in the root;2,4-DNP, 2,4-dinitrophenol.

14* 0031-9317/80/100199-09 $03.00/0 © 1980 Physiologia Plantarum

200 PAUL JENS6N AND ANDERS KYLIN Physiol. Plant. 50. 1980

Materials and methods

Germination and cultivation conditionsSeeds of spring wheat (Triticum aestivum L. cv. Svenno),oat (Avena .sativa L. cv. Brighton) and glasshousecucumber (Cucumis sativus L. cv. Bestseller Fl) weregerminated in Petri dishes on filter papers moistened withdistilled water at 20°C. After 3 days in darkness for springwheat and oat and 4 days for cucumber, the seedlingswere transferred to round plastic discs, 35 mm in diame-ter. Four plant groups, with 17 plants (spring wheat, oat)or five plants (cucumber) in each group, were grown to-gether for seven days in black-painted 2 1 glass beakerscontaining 1800 ml nutrient solution. The seedlings weregrown on complete nutrient solutions, which were dilu-tions (1, 25 and 50%) of a standard culture medium(=100%, see Kahr and Max Moller 1976) containing 6.0mM KNO,; 3.0 mM KH2PO4; 5.0 mM K2HPO4; 3.0 mMCa(NO,)2; 3.0 mM MgSO4; 4.0 mM Na2SO4; 3.0 mMNH4CI; 10.2nA/ Fe-EDTA; 10.0 \iM Mn;lnM Cu; 0.017HM Mo; 0.010 \iM Zn; 21.3 \iM B, pH 7.0. The initialconcentrations of K" and Ca " in the nutrient solutionswith different ionic strengths were in mAf: K"*, 0.19 (1 %);4.75 (25%); 9.5 (50%) and Ca "", 0.03 (1%); 0.75(25%); 1.5 (50%). The cultivation nutrient solutionswere radiolabelled with "''Rb (as a tracer for K"*") and " Cafrom the start. The specific activities were c. 20 jiCi/l for*"Rb and c. 40 ^Ci/l for "^Ca. The solutions werechanged day three and day six (on the day before theefflux experiments) and were continuously aerated. Wa-ter losses in the beakers were compensated for daily withdistilled water.

The plants were grown in climate chambers at a temp-erature of 25± r C and with continuous light provided byGeneral Electric F 48 power groove 17-CVX llOWfluorescent lamps (c, 25 W/m*). The relative humidityaround the shoots wasr. 50% during the cultivation. Theexception was that in half of the experiments - thosewhere efflux was done under high relative humidity — theplants were pretreated in a RH of r. 90%, 24 h prior tothe experiments.

Efjliix experimentsDuplicate experiments were independently performed foreach plant species in both 50% and 90% RH. The temp-erature and the light were the same as during cultivation.At an experiment two plant groups were simultaneouslyand successively transferred to a number of 1 1 glass beak-ers, each containing 800 ml continuously aerated un-labelled solution with the same composition as the sol-ution used for tracer loading. The radiolabelled ionseluted from the roots into the unlabelled solution weredetermined in the time intervals (in min): 0-5, 5-10,10-20, 20-^0, 40-50, 50-^0, 60-70, 70-80, 80-90,90-100, 100-110, 110-120, 120-180, 180-240,240-360, and 360-480. After the washout periods sam-ples of 3 ml were taken from each of the elution solutions.

At the end of an experiment the roots were gently blottedbetween filter papers. The plant groups were divided intoroots and shoots, which were weighed for fresh weightsand then digested by a mixture of HNO3 and HCIO4 involume proportions 2:1. The loss of radio-activity fromthe roots and that remaining in the plants were deter-mined by liquid scintillation spectroscopy. The K"*" andCa' "'" contents in the roots and shoots at the end of theexperiments were determined by chemical assay (atomicabsorption spectrophotometry).

Results

concentra-Fresh weights and A:+(*''Rb) and Ca^+Ctions in the rootsThe fresh weights of the roots and shoots of cucumberwere significantly higher than those of spring wheat andoat after a week in nutrient solutions with different ionicstrength (Table 1). However, the cucumber plants werecomparatively more inhibited in the 1 % nutrient solutionthan the other species. Moreover, the root/shoot freshweight ratio of cucumber was low and fairly constant,while it varied between 0.45 and 0.79 for wheat and oat,depending on the ionic strength of the cultivation sol-ution.

Figure 1 shows the concentrations of K'^(^''Rb) andCa^"^('*^Ca) at the start of the experiments. The concent-rations of the ions in the roots are calculated as the sum ofthe radioactivity of all the elution solutions plus theradioactive residue in the roots at the end of the experi-ment. The roots of all three species appear to be nearlysaturated with K'^(**'Rb) when grown in 25% nutrientsolution, since cultivation of the plants in 50% solutiononly gave a small further increase in [K"''(**Rb)]roo,. Incontrast, [Ca^"^(''*Ca)]root was linearly related to the ionicstrength of the solution, and thus to the external Ca '*'

Table 1. Fresh weight per root system and per shoot of glasshousecucutnber, oat and spring wheat. Plants cultivated during 7 daysin nutrient solutions, 1, 25 and 50% of a full solution (set to100%, for composition see Material and Methods). Values aremeans ± SD of four independent experiments.

Species

Cucumber

Oat

Spring wheat

% of afull •

nutrientsolution

12550

12550

12550

Fresh

Root

0.16±0.020.34±0.180.3110.19

0.1010.040.1510.050.1310.03

0.1110.020.1310.020.1410.02

weight, g

Shoot

0.67 + 0.051.6610.641.7010.71

0.1410.010.29+0.050.2810.03

0.1410.030.2910.070.2810.06

Root/shootfresh weight

ratio

0.240.200.18

0.710.520.46

0.79 .0.450.50

Physiol. Plant. 50. 1980 EFFLUX OF K+(«*"Rb) AND FROM ROOTS 201

OO

o

D)

OEZL

oo

uouI

OC

00

150

100

5 0

200

150

100

50

2 0 0

150

100

50

•I

A-

126 + 11

100 ^.^^^

-r57t10

" c175t18

/

81y'"-•^4618

- E181 i 12

/

/

/

5-«-37 + 3

100

1

139

1

144

1

100

1

196±3^-§-•— • ^132

1

2 0 6 1 6 , , ^

138

1

B

100

1

D

27

F

24

^ '

Y = 0.12X + 3.83

r = 0.996

Y = 0.19XH

r = 0.995

IT1 + O.3

Y = 0.17X -1r = 0.992

'^Xi ±0.2

100

— 1 ^

1

fO.64

73

"*!"*

1

^0.54

62

-"•-^V

1

9 . 9 ± 2 ^

.5+0.3

10.3 + 2^ ^

.810.8

9.1 + 2.'

^ - - ^.1 +0.8

100

-

1

1C1

-

1

92•1

1 T-

1 )

^ 1 5

10

5 2"o

0 i

? 15

10

5

0

15

10

5

ion

rat

c0)uc0u1

0U

CM

au

25 50 1 25!{) of a full strength nutrient solution

50

Figure 1. Concentration of K ^(^''Rb) (A, C, E) and Ca ^*(*^Ca) (B, D, F) in roots of glasshouse cucumber (A and B), oat (C and D) andspring wheat (E and F) at the start of the efflux experiments. Plants cultivated during seven days in ' ''Rb and "" Ca double labellednutrient solutions, 1, 25 and 50% of a full nutrient solution (= 100%). Each point represents the mean value of four plant groups inone experiment. Figures beside horizontal bars indicate mean values 1 SD of four (or three) independent experiments. Figures aboveor below vertical bars relate the concentration of K"^('"'Rb) and Ca-'^C^Ca) in the roots of oat and spring wheat to that in glasshousecucumber (set to 100% for each of the nutrient solutions used).

concentration as well. Correlation coefficients (r) of0.996, 0.995 and 0.992 were obtained for cucumber, oatand spring wheat respectively. It is noteworthy that[Ca ''"('**Ca)]root in spring wheat and oat was only about25% of that in cucumber after cultivation in the 1% nut-rient solution and that with increasing ionic strength ofthe solution [Ca ''"('*^Ca)]root gradually increased in wheatand oat in comparison with cucumber; so that it was ofsimilar magnitude in the roots of all three species whencultivation occurred in 50% nutrient solution.

K^(^^Rb)/K'^ selectivity in the roots and the shootsThe values for [Ca' '*']rooi obtained by chemical analysiswere consistent with those obtained by " Ca traceranalysis. However, the spectrophotometric determinationof [K" ]roo( did not agree with the **Rb analysis, except forhigh-K"*" roots of cucumber which were cultivated in 25%and 50% nutrient solutions (Table 2). Otherwise, the K*concentrations in the roots determined by **Rb analysiswere either strongly over- or underestimated dependent

on the K" status. K" was preferred against Rb"*" in low-K"*"roots of all three species, while Rb" was the preferred ionin high-K" roots of oat and spring wheat. The same hasbeen demonstrated for different varieties of barley byMarschner and Schimansky (1971) and by Pettersson(1978). Lack of significant discrimination between K"*"and Rb" (as in our high-K"^ roots of cucumber) has beenreported several times, e.^. by Lauchli and Epstein (1970)in excised roots of corn.

The K''(**Rb)/K'^ selectivity in the shoots was not gen-erally the same as in the roots (Table 2). The retention ofRb" was proportionally higher than that of K" in high-K"^roots of oat and wheat, whereas only little "extra" Rb*was transported to the shoots of the corresponding plants;thus indicating a differential K' (**'Rb)/K" selectivity inuptake, retention and transport (Pettersson 1978).

Analysis of the efflux dataA plot of the logarithm of the isotope remaining in theroot against time in unlabelled nutrient solution typically

202 PAUL JENSEN AND ANDERS KYLIN Physiol. Plant. 50. 1980

Table 2. K''('"'Rb)/K'^ selectivity in the roots and the shoots of seedlings of glasshouse cucumber, oat and spring wheat, each speciesgrown on three nutrient solutions with different ionic strengths. The K"'' concentration was determined after the efflux experiments by"••Rb tracer analysis and by atomic absorption spectrophotometry. Values are means of four independent experiments + SD. Eachexperiment represents the mean value of four plant groups analysed separately.

Ionic strength, 7o ofa full nutrient solution

/K''selectivity

Glasshousecucumber

Oat Spring wheat

12550

Root

0.6110.0.99 + 0.0.9710.

129103

Shoot

0.42 + 0.0.9110.0.93 + 0.

130703

0.1.1.

Root

7810.74+0.7610.

120915

0.1.1.

Shoot

60+0.1010.14+0.

140812

011

Root

.52+0.

.6010.

.64 + 0.

070105

011

Shoot

.60+0.

.07+0.

.19+0.

150810

gives curves (Figure 2, curves (a)), which can be resolvedinto linear phases corresponding to three cellular com-partments—cell wall, cytoplasm and vacuole. The kineticsof efflux is often explained according to a model with thecell compartments in series, by the compartment analysismethod first described by Pitman (1963). The method hasbeen used for the analysis of the present data, althoughsome of the elution curves obtained for both K' C^Rb)and Ca^* had "shoulders" (Figure 2, curves (b) and Table3). The irregularity appeared in the same time interval (c.20—120 min) for both ions. "Shoulders" in the wash-

ing-out curves were more frequent for Ca^'''(''^Ca) thanfor K+C' Rb) in 25% and 50% nutrient solution (Table3).

and Ca +C^^Ca) efflux from the different rootcompartments (cell walls, cytoplasm and vacuoles) at dif-ferent ionic strengths of the mediumDue to the irregularities in the efflux curves, a practicalapproach has been taken in the handling of the data, sothat fixed times (10 and 180 min, respectively) have beenallotted for "complete leakage" from the cytoplasmic and

oo

oo

o5

- 3.00 w

- 2.50

- 2.00

- 1.50

- 1.00

- 0.50

- 0.50

120 2 4 0 360 480 0 120

Elution time, min2 4 0 3 6 0 4 8 0

CT)

OO

CT)

CTEo

aU

in

CMO

u"oE

Figure 2. Time course of efflux of K*(^Rb) (A) and Ca^*(*^a) (B) from roots of intact cucumber plants to unlabelled nutrient solution.Typical examples of (a) continuous elution curves in 1% nutrient solution; and (b) irregular elution curves in 50% nutrient solution.The elution solution was changed as indicated by the data points. RH was 50%.

Physiol. Plant. 50. 1980 EFFLUX OF K+(»''Rb) AND FROM ROOTS 203

Table 3. Continuous (+) and irregular (-) elution curves for K'^(^^Rb) and Ca^'^(*^Ca) in theexperiments of cucumber, oat and wheat, at different ionic strengths of the nutrient medium. Eachsymbol represents one experiment. ND = not determined. The number of experiments showingcontinuous and irregular elution curves for each ion, are summarized at bottom of the Table.

Plant species RH%

Ionic strength,% of a fuUstrength nutrient solution

25 50

Cucumber

Oat

Spring wheat

50905090

5090

+ 4-

+ ++ +

+ -1-

-1- +

- N D - N D

ContinuousIrregular

12 +0 -

120

10 + 2 +2 - 10 -

7 + 2 +4 - 9 -

30

20

C 10oo

— 20

o£^ 10o3*

s. 20

p

100

80

6 0

oo

100 —

80

60

oE

o

100

80

6 0

1 25 50 1 25 50

% of a full strength nutrient solution

raU

Figure 3. Total efflux of K *(^''Rb) and Ca^*(*^Ca) from the rootsof glasshouse cucumber (A and B), oat (C and D) and springwheat (E and F) in the time interval 0-480 min in inactive nutrientsolutions of three different ionic strengths and with two relativehumidities around the shoots, 50% (D) and 90% (41. Valuesexpressed in % of the concentration of the ions in the roots at thestart of the experiments. Each single experiments is a mean offour plant groups. Except where vertical bars are lacking, eachcolumn is a mean of two independent experiments 1 SD. Figuresabove black columns relate efflux from roots of plants in 90%RH to those of plants in 50% RH (set to 100%).

from the wall compartments. These times have beenchosen after compartment analysis of the smooth effluxcurves.

The pattern for total efflux from the roots after 8 h wassimilar for the species, but was different for K ' ( ''Rb) and

(Figure 3). Between 55 and 100% of the

10

I 5oo „

10

1

100

f l_88u

O40 -

20

o0 E

60 •r

40 ^"53

20 T,

40 r<u

20

1 25 50 1 25 50% of a full strength nutr ient solution

raO

Figure 4. Efflux of K*(^''Rb) and Ca^*(*^Ca) from the cell wallsof the roots in the time interval 0-10 min. Values expressed in %of the concentration of the ions in the roots at the start of theexperiments. Symbols and statistics as in Figure 2.

204 PAUL JENSEN AND ANDERS KYLIN Physiol. Plant. 50. 1980

initially present in the roots was washed outduring the 8 h of elution, while the efflux of K+(**'Rb)only was between 10 and 25%. In cucumber and wheat,the efflux of K''"('"'Rb) increased with increasing ionicstrength of the root medium, especially at 50% relativehumidity. The flux of K' (*"'Rb) out of the roots duringconditions with high rate of transpiration (50% RH) washigher than under conditions with low rate of transpira-tion (RH 90%), except for oat and wheat in 1% solution.Contrariwise, there was no obvious systematic influenceof relative humidity on the efflux of Ca ' C' Ca). Theefflux of K"^('"'Rb) was highest in 50% nutrient solutionwhere efflux of Ca ' C^Ca) was lowest. The Ca ' C^Ca)efflux was noticeably higher from cucumber and wheatroots than from oat in 1 %> nutrient solution.

As for efflux from the cell walls of the roots (Figure 4),alt three species behaved more or less in the same way.Although the concentration of Ca "'"('**Ca) in the roots ofcucumber was about four times that in the other twospecies at the lowest ionic strength (Figure 1), the effluxof Ca' *(*'*Ca) from the cell walls consitituted a constantpercentage (c. 20%) when calculated with the initial

concentration as a basis. The highest values

o

oE3.

O

•I

ra 2ao

Eo~ 6

143

8 0

60 'i

4 0

2 0

6 0

4 0

2 0

6 0

40

20

oE

a.o

"(0

O

1 25 50 1 25 50% of a full strength nutrient solution

Figure 5. £//7u.v of K *(^Rb) and Ca ^*{*H:a) from the cytoplasmof the roots in the time interval 0-180 min. Values expressed in %of the concentration of the ions in the roots at the start of theexperiments. Symbols and statistics as m Figure 2.

•r 0

3*

•= 2

30

20

10

0 £

15 o

10

20

<nao3Ora>Eo

raU

10 £ ,

toO

1 25 50 1 25 50

% of a full strength nutrient solution

Figure 6. Efflux of K*(^Rb) and Ca^*(*^Ca) from the vacuoles ofthe roots in the litne interval 0—480 min. Values expressed in % ofthe concentration of the ions in the roots at the start of theexperiments. Symbols and statistics as in Figure 2.

for the cell wall were in all cases obtained in the 25%nutrient solution. There may be an effect of the transpi-ration rate on the K" (*''Rb) efflux, but not on theCa'-'C^Ca) efflux, from the cell walls of the roots. Thesignificant differences in total K' (®*'Rb) efflux betweenplants with different transpiration rate (Figure 3) cannot,however, be explained to more than a minor extent bydifferent efflux from the cell walls.

The pattern for Ca ' C^Ca) efflux from the cytoplasmicpart of the root cells was different from the efflux fromthe cell walls (Figure 5), In contrast to the efflux ofK''(*''Rb), the Ca^+C^Ca) efflux appeared to decreasewith increasing ionic strength of the nutrient solution (ex-cept for oat between 25 and 50%), while [Ca ''('*^Ca)]rootincreased linearly (Figure 1). A comparatively great partof the total efflux of Ca " appeared attributable to thecytoplasm. For K"*", high RH decreased the cytoplasmefflux all over, except for oat and wheat at the lowestionic strength.

The pattern for K+(»<'Rb) and Ca2+('**Ca) efflux fromthe vacuoles of the roots (Figure 6) was partly differentfrom that of the cytoplasm. As compared to cell walls and

Physiol. Plant. 50. 1980 EFFLUX OF K+(««'Rb) AND FROM ROOTS 205

Table 4. K-^(^''Rb) and Co^+C'Co) efflux from the roots of springwheat at 50% and 90% RH and in the absence and presence of2,4-DNP. Cultivation and efflux experiments on 25% of fullnutrient solution. 2,4-DNP was added to the unlabelled washsolution in a concentration of lO'^M. The ion efflux at 50% RHand in the absence of DNP during 480 min is set to 100% (con-trols), which corresponds to 45.3 nmol-g"' fr. wt of root forK+(»*'Rb) and 2.7 pimol-g" fr. wt of root for Ca^-'

Ion RH%

DNP in thesolution

Ion efflux, in the timeinterval 0^80 min

as % of control

50509090

50509090

- (controls)

- (controls)

100676660

10010192112

1005765-

10011395

cytoplasm, only a small fraction of the Ca ''"(''*Ca) initiallypresent in this compartment appeared to be eluted fromthe roots. In the main, high RH depressed the efflux ofK" (®*Rb) from the vacuoles, just as for the cytoplasm.

In young plants, 2,4-DNP in the nutrient mediumgreatly inhibits the influx of different ions, e.g. K' (®''Rb)and SO4^" (Pettersson 1966 a, b, Pitman and Saddler1967, Jensen 1978). Moreover, 2,4-DNP is taken up intothe roots and translocated to the shoots (Barber andKoontz 1963). Accordingly, addition of DNP to the nut-rient solution may throw light on the nature of the coupl-ing between the K'''(**'Rb) efflux and metabolism in theseexperiments. In Table 4 the effect of 10" M DNP isshown on the K' (**Rb) and Ca ' ('*^Ca) efflux from theroots of spring wheat in the 25% nutrient solution. DNPdid not affect the Ca^'^^^Ca) efflux, while the efflux ofK' (*'*'Rb) was inhibited 30^0% at 50% RH. Increase inhumidity inhibited the K" (*''Rb) efflux to the same extentas 2,4-DNP, and there was no further effect of DNP un-der high humidity conditions.

Discussion

Most investigators studying the processes of efflux haveused excised plant tissues as experimental material, e,g,slices of beet (Pitman 1963), discs of red beet (Poole1969), ve«a coleoptiles (Pierce and Higinbotham 1970)and excised segments of Pisum sativum roots (Etherton1967). The elution curves obtained were continuous andinterpreted according to the compartmental analysismethod. An irregular pattern of K' (**Rb) efflux was re-ported from excised root segments of maize seedlings atlow external concentrations of KCl (Pallaghy et al, 1970),although the anomaly was not found when the K" (*^Rb)efflux was determined from the excised roots at high am-bient K" concentrations (>1 mM), or in the roots of in-

tact low-K"*" plants. The latter observation is consistentwith the present results (Table 3). Contrary to the find-ings of Pallaghy er fl/. (1970), the present experiments andexperiments by Erlandsson (1979) with wheat show ir-regularities in the elution curves at high external ion con-centrations when intact plants are used.

The parallel pathway model suggested by Pallaghy et al.(1970) to explain the "shoulder" observed in the elutioncurve differs from the three-compartments-in-seriesmodel (Pitman 1963) by having an additional compart-ment in the cytoplasm. Since it was the special aim of ourstudy to compare fluxes of K" (**'Rb) and Ca "*" out of themain cell compartment in the roots of three plant sepcies,appropriate time intervals were selected from the threehnear phases obtained by compartment analysis of thesmooth elution curves at low ionic strength. The sametime intervals were assumed to hold in the evaluation ofthe deformed elution curves. The time intervals agreerather well with those found by other workers, e.g. byPierce and Higinbotham (1970), who studied the loss ofK"*", Na"*" and Cl~ from different compartments of coleop-tile cells of oat.

With regard to the "irregular" efflux reported in Figure2 and Table 3, one possibility was that the irregularitiesmight be coupled to the transpiration stream of water,which could conceivably carry some of the leaking ionsinwards and so complicate the pattern of ions given off tothe medium. However, this possibility appears of littleimportance for the shoulders, since we found ir-regularities in the elution curves also during conditionsgiving low transpiration at high external ion concentra-tions. In the lowest ionic strength, the regular elutioncurve for the ions was always obtained in both relativehumidities.

The K"'(*''Rb)/K+ selectivity in the roots (Table 2)does not vary in the same way for the plant species com-pared. However, insignificant differences in selectivity forK" and Rb"*' in the shoots were obtained between thespecies at all three nutrient strengths tested (Table 2).The K' (**'Rb)/K' selectivity in the shoots was close tounity after cultivation in the two nutrient solutions of25% and 50% ionic strength. Thus there is no discrimi-nation between K" and Rb" in the transport from roots toshoots of high-K"* plants. It can be argued that the samecondition should hold for flux of K" and ***'Rb out of theroots and to the medium. If so only efflux of K"*" fromlow-K"*" roots ought to be corrected. This has not beendone, however, since K'''(**'Rb) efflux has been related tothe initial concentration of K" ('*''Rb) in the roots at thebeginning of the experiment (Figures 3—6). Moreover, thevarying discrimination between K" and Rb"* cannot affectwhat appears to be the main conclusions of our work,those about an influence of RH on efflux from the cyto-plasm and vacuole of the root cells. A discrimination be-tween K and Rb varying with concentration of K"*" wasnoted and discussed by Jacoby and Nissen (1977).

There is an additional aspect of the different selectivity

206 PAUL JENSEN AND ANDERS KYLIN Physiol, Plant. 50, 1980

of roots and shoots towards K'*' and Rb"*". Under condi-tions where the roots took up Rb"* preferentially (oat andwheat in 25 and 50% media), there was little discrimina-tion between Rb" and K* in the transport to the shoots.Under conditions where the roots took up K"*" preferen-tially or discriminated only little between Rb"*" and K"*",the shoots more or less followed the roots. The loadingtime in the experiments (one week) was long as comparedto the wash-out time (8 h). Thus, the different discrimi-nation of shoots and roots between Rb" and K" shouldprobably be ascribed to the loading period and be littleaffected by transport from the root vacuoles to the shootor by retranslocation from the shoot to the roots duringwash-out (cf. Anderson 1975. whose reservation cannotapply here). The transport processes involved are then(LCittge 1974): a) uptake through the the plasmalemma ofthe cortical root cells, b) accumulation through the to-noplast of the cortical root cells into the vacuoles, and c)transcellular translocation through the symplasm andexcretion into the xylem. Since process (a) is the same forroots and shoots, the different abilities to discriminatebetween Rb" and K" must be coupled to differences be-tween processes (b) and (c); and the system that variesthe most in its discriminatory properties must be the to-noplast of the cortical root cells (Table 2).

The variations in K'*'(*"'Rb) and Ca""*" concentrations inthe roots (Figure 1) may indicate both quantitative andqualitative differences in ion accumulation between theplant species as well as between K^ and Ca*" . Differentmechanisms regulate the ion concentrations in the roots.Influx of K*(Rb'* ) into the root cells is suggested to beboth metabolic and non-metabolic, but the proportionbetween the two components differs, due to nutrientstatus (Pettersson and Jensen 1979) and age of the plant(Jensen 1978). At high K" status ofthe roots, as obtainedafter cultivation in 25% and 50% nutrient solutions, in-flux of K" (^''Rb) is probably strongly allosterically inhi-bited (Glass 1976, Jensen and Pettersson 1978). whilethis type of regulation may not exist for Ca'"* . An activemechanism for Ca** was, however, reported by Dunlop(1973). Non-metabolic Ca"'*' uptake has been suggestedto occur into algal cells (Kesseler 1964, Goldman etal,1972). At the same time, it is not clear whether the lowelectrochemical activity of Ca"'' in algal cells is due to lowpermeability alone or requires an active efflux (Walker1957, Spanswick and Williams 1965).

Efflux of Ca "* from roots has been little studied. In-stead, the effects of Ca^* on the efflux of other ions hasbeen discussed. The efflux of ions from certain algal tis-sues and the roots of barley and maize is much faster inthe absence of Ca "* than in its presence (Elzam and Ep-stein 1965, Maas and Leggett 1968, Weigl 1969). How-ever, there was a constant ionic balance in our experi-ments. Clarkson (1974) pointed out that the externalCa-* concentration must exceed lO^Af, since normalmembrane function seems to depend strongly on thepresence of Ca^*, and possibly the low external Ca^* con-

centration (0.03 mAf) in the 1% solutions causes mem-brane defects. This might be indicated by the high pro-portion of Ca ' flowing from the cytoplasm at the lowestionic strength of the solution, especially from cucumber(Figure 5). Growth was severely reduced for all threeplant species in 1 % solution (Table 1).

The main part of the Ca '''('**Ca) initially present in theroots was exchanged during the course of the experimentsin 1% and 25% of the full nutrient solution (Figure 3).The proportion of Ca ' in the rapid fraction (Figure 4)was higher in 25% medium than in 1% or 50%. Thereason for this is obscure, but logically it appears that atleast two different functions must be involved, for in-stance the adsorption isotherm for the cell wall space andan active outpump as demonstrated by Maeklon (1975)for roots of AUium,

As compared to high humidity (90% RH) around theshoots during the efflux period and the day before, lowhumidity (50% RH) increased efflux of K' (»*'Rb) (Fig-ure 3). The compartmental analysis (Figures 4—6) showsthat the effect is due to the cytoplasmic and vacuolarcomponents, so that it is probably localized in the plas-malemma and tonoplast. Thus, there must be a regulatorysystem from the shoots that is triggered by "drought"conditions and signals to the root cells to increase leakage— a signal that should reasonably lead to increased trans-port of ions also with the water stream delivered to thexylem. This reaction may in the final end give the shootcells possibilities to increase their supply of ions andmaintain a water potential low enough to meet theoriginal "drought" signal. — It is interesting to note thatanother "drought" signal, namely a decreased water po-tential (-5 to -10 bar as polyethyleneglycol) also leads toincreased potassium efflux, presumably through an effecton the permeability of the root cell membranes for potas-sium (Erlandsson 1979). An effect of decreasing waterpotential was not seen in our material and can hardly beexpected in a clear form, first since even the strongestsolution has a water potential in the order of only -0.07bar and secondly because the osmotic effect must in ourcase be combined with the influence of the specific ions.

The high efflux of K''(*"'Rb) under "drought" condi-tions was inhibited by DNP (Table 4) to about the sameextent as by high air humidity. The effect of high airhumidity was not increased by DNP and vice versa. Pet-tersson (1966a,b) and Christersson and Pettersson (1968)gave evidence that the rate of the water stream throughthe roots can affect the turnover of sites for sulphatetransport and that these sulphate sites are inhibited byDNP. A corresponding mechanism for potassium trans-port may be indicated as part of the chain between shootsand roots in our experiments.

We thank Professor Sune Pettersson for coordinating our effortsat the start of the investigation. Mrs Inger Rhodin and MrsAnn-Christine Holmstrom provided skilful technical assistance.

Physiol. Platit. 50. 1980 EFFLUX OF K+(«''Rb) AND FROM ROOTS 207

and Mrs Lena Strandh turned our notes into a readable manu-script. The work was supported by grants from the SwedishNatural Science Research Council to one of us (Anders Kylin).

ReferencesAnderson, W. P. 1975. Long distance transport in roots. -In Ion

Transport in Plant Cells and Tissues (D. A. Baker and J. L.Hall, eds.), pp. 231-266. - North Holland Publishing Com-pany, ISBN 0-7204-4519-1.

Barber, D. A. & Koontz, H. V. 1963. Uptake of dinitrophenoland its effect on transpiration and calcium accumulation inbarley seedlings. - Plant Physiol. 38: 60-65.

Christersson, L. & Pettersson, S. 1968. Water and sulphate up-take at root reduction in Ricinus communis. - Physiol. Plant.21: 414-422.

Clarkson, D. T. 1974. Ion Transport and Cell Structure in Plants.- McGraw Hill, Maidenhead, U.K. ISBN 07-084026-1.

Dunlop, J. 1973. The kinetics of calcium uptake by roots. -Planta (Bed.) 112: 159-167.

Elzam, O. E. & Epstein, E. 1965. Absorption of chloride bybarley roots: kinetics and selectivity. - Plant Physiol. 40:620-624.

Erlandsson, G. 1979. Efflux of potassium from wheat roots in-duced by changes in the water potential of the root medium. -Physiol. Plant. 47: 1-6.

Etherton, B. 1967. Steady state sodium and rubidium effluxes inPisum sativum roots. - Plant Physiol. 42: 685-690.

Glass, A. D. M. 1976. Regulation of potassium absorption inbarley roots. An allosteric model -Ibid. 58: 33-37.

Goldman, J. C, Porcella, D. B., Middlebrooks, E. J. & Toering,D. F. 1972. The effect of carbon on algal growth - its re-lationship to eutrophication. - Water Res. 6: 637—679.

Jacoby, B. & Nissen, P. 1977. Potassium and rubidium interac-tion in their absorption by bean leaf slices. - Physiol. Plant.40: 42-^4.

Jensen, P. 1978. Changes in ion transport in spring wheat duringontogenesis. -Ibid. 43: 129-135.

- & Pettersson, S. 1978. Allosteric regulation of potassium up-take in plant roots. -Ibid. 42: 207-213.

Kahr, M. & Max Moller, I. 1976. Temperature response andeffect of Ca " and Mg " on ATPases from roots of oats andwheat as influenced by growth temperature and nutritionalstatus. -Ibid. 38: 153-158.

- Bervaes, J., Kylin, A. & Kuiper, P. J. C. 1977. Influence ofmineral nutrition on ATPase activities and relation of satu-rated to unsaturated fatty acids in roots of wheat and oats. -InTransmembrane Ionic Exchanges in Plants. (M. Thellier er al.,eds.), pp. 213-217. Colloque du C.N.R.S. n:o 258 (1976).ISBN 2-222-02021-2.

Kawasaki, T., Kahr, M. & Kylin, A. 1979. Interactions of dival-ent cations in their action on ATPases from cucumber roots. -Physiol. Plant. 45: 437^39.

Kesseler, H. 1964. Collection of cell sap, apparent free space andvacuole concentrations of the osmotically most importantmineral components of some Helgoland marine algae. - Hel-golander Wiss. Meeresunters. 11: 258—269.

Lauchli, A. & Epstein, E. 1970. Transport of potassium andrubidium in plant roots. The significance of calcium. - PlantPhysiol. 45: 639-641.

Luttge, U. 1974. Cooperation of organs in intact higher plants: areview. -In Membrane Transport in Plants (U. Zimmermannand J. Dainty, eds.), pp. 353-362. - Springer-Verlag. ISBN3-540-06989-5.

Maas, E. V. & Leggett, J. E. 1968. Uptake of ""Rb and K byexcised maize roots. - Plant Physiol. 43: 2054-2056.

Macklon, A. E. S. 1975. Cortical cell fluxes and transport to thestele in excised root segments of Allium cepa L. II Calcium. —Planta (Berl.) 122: 131-141.

Marschner, H. & Schimansky, C. 1971. Suitability of usingrubidium-86 as a tracer for potassium in studying potassiumuptake by barley plants. - Z. Pflanzenernahr. Bodenkd. 128:129-143.

Pallaghy, C. K., Luttge, U. & von Willert, K. 1970. Cytoplasmiccompartmentation and parallel pathways of ion uptake inplant root cells. — Z. Pflanzenphysiol. 62: 51—57.

Pettersson, S. 1966a. Active and passive components of sulphateuptake in sunflower plants. - Physiol. Plant. 19: 459^92.

- 1966b. Artificially induced water and sulphate transportthrough sunflower roots. —Ibid. 19: 581—601.

- 1978. Varietal differences in rubidium uptake efficiency ofbarley roots. —Ibid. 44: 1—6.

- & Jensen, P. 1979. Regulation of rubidium uptake insunflower roots. -Ibid. 45: 83—87.

Pierce, W. S. & Higinbotham, N. 1970. Compartments andfluxes of K" , Na" , and Cl" in Avena coleoptile cells. - PlantPhysiol. 46: 666-673.

Pitman, M. G. 1963. The determination of the salt relations ofthe cytoplasmic phase in cells of beetroot tissue. - Aust. J.Biol. Sci. 16: 647-668.

- & Saddler, H. D. 1967. Active sodium and potassium trans-port in cells of barley roots. -Proc. Natl. Acad. Sci. U.S.A. 57:44-^9.

Poole, R. 1969. Carrier mediated potassium efflux across the cellmembrane of red beet. - Plant Physiol. 44: 735-739.

Spanswick, R. M. & Williams, E. J. 1965. Ca fluxes and mem-brane potentials in Nitella translucens. - J. Exp. Bot. 16:463-473.

Walker, N. A. 1957. Ion permeability of the plasmalemma of theplant cell. - Nature (Lond.) 180: 94-95.

Weigl, J. 1969. Efflux und Transport von Cl" und Rb" in Mais-wurzeln. Wirkung von Aussenkonzentration, Ca"*""*", EDTAund lES. - Planta (Berl.) 84: 311-323.

Edited by SP.