Involvement of Proton Fluxes in the K'-Na' Selectivity at the Plasmalemma; K'-dependent Net Extrusion of Sodium in Barley Roots and the Effect of Anions and pH on Sodium Fluxes

Lehrstuhl fUr Botanik I, Universitat Wiirzburg, Federal Republic of Germany

Involvement of Proton Fluxes in the K+-Na+ Selectivity at the Plasmalemma; K+-dependent Net Extrusion of Sodium in Barley Roots and the Effect of Anions and pH on Sodium Fluxes WOLF DIETER JESCHKE

With 8 figures Received October 10, 1979 . Accepted December 10, 1979

Summary Using excised, Na+-Ioaded barley (Hordeum distichon) roots, fluxes of sodium at the cellular membranes and cytoplasmic and vacuolar sodium contents were studied as a function of accompaying anions and the pH. Na+ fluxes and particularly transport of sodium across the roots were substantially decreased (when the anion was varied) in the order NO s- > CI- ~ 504-- but the vacuolar content and tonoplast fluxes were less affected. In the presence of any anions potassium induced a transient net sodium extrusion across the plasmalemma and selective inhibition of Na+ transport, again the effect being smallest in the presence of 504--' Similarly, all Na+ fluxes were reduced when the pH was decreased and K+ induced a net Na+ extrusion at all pH values. When related to the cytoplasmic Na+ content, the highest relative rate of Na+ extrusion was found at low pH. Net Na+ extrusion across the plasmalemma could be induced also by an increase in [H+] from pH 8 to 4. This H+-dependent Na+ extrusion was not transient and was followed by increased vacuolar Na+ efflux; it did not result in selective inhibition of trans-root Na+ transport. The H+-dependent Na+ extrusion was smaller than K+-dependent one and was greatly stimulated when potassium was added simultaneously with acidification from pH 8 to 4, thus pH 4 does not inhibit K+-Na+ exchange. The results are consistent with a mediation of K+-Na+ selectivity at the plasmalemma by proton fluxes. K+ stimulated the Na+ efflux also when added subsequent to an acidification and then brought about a reversal of the proton-induced enhancement of vacuolar Na+ efflux. This indicates an essential role of potassium ions in the regulation of cytoplasmic pH. Key words: K+-Na+ selectivity, proton fluxes, sodium fluxes, effect of anions, effect of pH, plasmalemma, barley roots.

Introduction K+-Na+ selectivity in barley roots is in part due to an active efflux of sodium and SADDLER, 1967) that is linked to an influx of potassium (JESCHKE,

(PITMAN

Dedicated to Professor Dr. WILHELM SIMONIS on the occasion of his 70th birthday.

Z. Pflanzenphysiol. Bd. 98. S. 155-175. 1980.

156

WOLF DIETER JESCHKE

1973). The stoichiometry of this K+-Na+ exchange is not 1 : 1 hut varies, suggesting the fluxes to he indirectly coupled possibly by means of proton fluxes (JESCHKE, 1977 h). There is much evidence to suPPOrt K+/H+ exchange at the plasmalemma (JACKSON and ADAMS, 1963; PITMAN, 1970; MARRE, 1977) powered hy electrogenic proton extrusion (MITCHELL, 1966; RAVEN and SMITH, 1977; POOLE, 1978). The proton motive force Ap, that is the sum of a membrane potential difference, Alp and the pH gradient, ApH, may power other fluxes such as anion influxes or cation effluxes (SMITH, 1970; SLAYMAN, 1974). Models for the coupling of Na+ efflux to proton influx have been presented for bacteria (HAROLD and PAPINEAU, 1972; WAGNER et aL, 1978) and fungi (SLAYMAN, 1974). An involvement of a proton pump has been proposed also for higher plants (HODGES, 1973; RATNER and JACOBY, 1976). The latter presented a scheme similar to that for bacteria. Their findings differed from our previous ones in that K+-Na+ exchange was dependent on the presence of S04-- - inducing net H+ efflux (RATNER and JACOBY, 1976) while in differentiated barley roots a massive K+-Na+ exchange is possible in the pl1esence of NO s- (JESCHKE, 1973; 1977 a-c; JESCHKE and STELTER,1973). In the present paper, therefore, the effect of anions and of protons on K+-Na+ exchange in differentiated barley roots was investigated. The results support the involvement of protons as suggested hy RATNER and JACOBY (1976) and further show proton-mediated K+-Na+ exchange to occur also when there is no net H+ efflux. The data suggest an essential role for potassium in the regulation of the cytoplasmic pH (RAVEN and SMITH, 1977). Part of the present results have been reported at the symposion of the European Phytochemical Society in Bangor, April 1978 (JESCHKE, 1979 b). Material and Methods 1. Abbreviations influx and efflux at the plasmalemma of the cortical cells [pmol g-Ifr.wt. h- I ], as for the following fluxes or transport rates. influx and efflux at the tonoplast. 0 CY' "ye: R': rate of transport of ions to the xylem exudate. cytoplasmic and vacuolar ion content [,umol g-Ifr.wt.]. Qe' Qv: specific activity [cpm ,umol- I] of tracer 22Na in the cytoplasm, vacuole or the xylem exudate.
"'oe' "'co:

Plants. Barley seedlings, Hordeum distichon L., C.v. «Kocherperle», were grown for 4 days in the green house in 0.5 mM CaS04 solution. The roots - «low-salt-roots» - were about 5 cm long, when harvested, and contained 8-10 ,umoles Na+ and 30 ,umoles K+ per gram fresh weight. Z. Pjlanzenphysiol. Bd. 98. S. 155-175. 1980.

Involvement of protons in K+-Na+ selectivity

157

2. Measurement of sodium fluxes

Unidirectional fluxes of sodium (22Na) were determined by efflux measurements and compartmental analysis (PITMAN, 1971). The methods have been described in detail previously (JESCHKE, 1977 b). In summary, excised low-salt roots were mounted in vessels containing two chambers (see insert in Fig. 1). The major (2.5-3 cm) part of the roots and the tips were in chamber «5», the cut ends protruded into the smaller chamber «X». The flux experiments generally included three periods, see Table 1. During the loading period (1. in Table 1) the roots were equilibrated with Na+ and 22Na+ in the absence of K+. During equilibration the transport of Na+ and 22Na to the xylem exudate (chamber X) and the specific activity of 22Na in the exudate (see below) were measured. In the subsequent period of elution (2. in Table 1) the roots were washed out with frequently changed volumes of unlabelled solutions. In the third period of «re-elution» (3. in Table 1) the washing-out Table 1: Composition of experimental solutions and measurable parameters in transport and efflux experiments. The present example refers to measurements at pH 8. experimental period, duration

1. loading 22h

2. elution,

6h

3. re-elution, 3-4 h

solution, volume

chamber 5':') measurements

basal medium (3 mM CaCI 2) 1 mM NaCl + 0.1 ,uCi 22Na/,umol pH 8'f~'), 100ml see'f)

net uptake of Na+ and 22Na; apparent':":') proton fluxes

basal medium + 1 mM NaCI pH 8'f'f), 20.5 ml frequently changed

efflux of 22Na; apparent'f':') proton fluxes

basal medium + 1 mMNaCI + 0.2mMKCI pH 8':'~') or pH 4':'~'*), 20.5 ml frequently changed

K+-dependent (net) sodium 2Na) efflux; net K+ uptake; apparent':'"') proton fluxes

+

solution, volume

chamber X'f) measurements

basal medium (3 mM CaCI 2),

9.5 ml repeatedly changed

e

basal medium, 9.5 ml frequently changed basal medium,

9.5 ml frequently changed

transport of Na+ and 22Na across the roots; specific activity Sx of xylem exudate transport of N a+ across the roots and of 22Na out of the roots; Sx

K+-induced inhibition of Na+ and 22Na transport; net transport of K+; Sx

,:.) For the chambers S and X see the insert in Fig. 1. During loading chamber 5 was connected to a larger reservoir, the solution was circulated by means of a peristaltic pump. 'f'f) The pH was maintained at the desired value either by buffers or by continuous titration (Methods). In this case the apparent proton fluxes were obtained from the acid or base consumption. *'f'f) In some experiments a jump in pH was induced by changing the pH from 8 to 4. Z. Pflanzenphysiol. Bd. 98. S. 155-175. 1980.

158

WOLF DIETER JESCHKE

procedure was continued but now with altered conditions i.e. with an addition of K+ or at lower pH, see below and Table l. Throughout the experiments the solutions in S and X were stirred by a stream of air or (at alkaline pH) of a mixture of 80 Ofo N 2 and 20 Ofo 02' The experiments were run at room temperature. At the end of each experiment the roots were weighed and their ,,-radiation was counted. They were then dried and ashed, the residue was dissolved in 0.1 mM HCI for K+ and Na+ analysis. The wash-out solutions were analysed for radioactivity and Na+ and K+ concentrations. In Table 1 the possible types of measurements, that were conducted during each experiment using the solutions collected from the chamber S or X are summarized. 3. Experimental solutions Although solutions of various composltIons were used to pursue -different experimental objectives, all contained a basal medium of Ca++ (3 mM) and sometimes Mg++ (0.5 mM) salts (For detailed information see Table 1 and legends of Figures). 4. Control of pH and measurement of apparent proton fluxes The pH was maintained at a constant value by (a) buffering, at pH 5.8 with 1 mM NaH2P04/Na2HP04' at pH 4 with 1 mM succinic acid adjusted to pH 4 with Ca(OH)/) or, at pH 8 with 1 mM HEPES/Ca(OHh; or (b) by titration, using a glass electrode in chamber S, a pH meter in combination with a titrator, a mechanical burette and a recorder (Radiometer A/S, Copenhagen). In this way the pH was continuously adjusted to 4 with HCI or to 8 with Ca(OH)/). The acid or base consumption was monitored and this permitted the apparent proton or hydroxyl ion fluxes to be measured. When working at pH 8 the vessels were carefully sealed and a mixture of 80 Ofo N 2 20 Ofo O 2 was used for aeration in order to avoid absorption of CO 2 that would alter the pH and mimic a proton excretion.

+

5. Analytical procedures 22Na radioactivity was measured using a ,,-scintillation-counter (Laboratorium Bethold, Wildbad, Schwarzwald, W. Germany). Concentrations of Na+ and K+ were determined with a flame photometer (Type «Eppendorf., Netheler and Hinz Comp., Hamburg, W. Germany). 6. Estimation of unidirectional fluxes and content of sodium The 22Na radioactivity in the wash-out solutions yielded the tracer efflux across the root cortex iP* out(S) and the transport of tracer to the xylem exudate iP* out(X). These tracer fluxes and the residual radioactivity in the roots were used to estimate sodium fluxes, the Na+ content, and the transport of Na+ across the roots as described in detail previously (JESCHKE, 1972, 1977b). The analytical data were processed in an off-line Wang 2200 S computer system in combination with a 2202 plotting output writer (Wang Laboratories, GmbH, Frankfurt, W. Germany). A program in BASIC language for evaluating the data has been developed in cooperation with Mr. Rudolf Behl. All fluxes and contents were related to the fresh weight of that part of the roots that was in the chamber S. 7. Measurement of transport and specific activity of sodium in the xylem exudate Since the medium in chamber X for collecting the xylem exudate initially was free of K+ and Na+ (Table 1), chemical analysis of these elements in the solutions collected in this chamber allowed the net transport of K+ and Na+ across the roots to be determined. From these data and the ra-dioactivity data, the specific activity of 22Na in the xylem exudate, sx'

1) Ca(OH)2 was used since NaOH or KOH would interfere with the measurements by changing the specific activity of 22Na or the Na+ fluxes respectively and since an excess of Ca++ was present at any rate. Z. Pflanzenphysiol. Bd. 98. S. 155-175. 1980.

Involvement of protons in K+-Na+ selectivity

159

was determined (see Fig. 1). A significant effect of omitting Na+ from chamber X on the transport of Na+ and 22Na to the xylem exudate could not be detected in comparative measurements, probably because very little root tissue protrudes into the collecting mamber X.

o • Q.1 m'1 KCt (,n

6x 0' ~

SI

A

2

_

x 0'

Sx

---------

0,6

R' 2 - ....- - - - - - ...

~ 07

::l E o

..

.c:

Eo .. 01

a 6

v-~

000

"::> E <: "l'"

-..g~

'Qj8~

"

o .. _

.. -:: 2 0 t::

eu

~

2

,,,

~\

, ,, FS

I

I

,, ,

~co

8

c

1\

:t

100

200

J()()

@

500 600 lIme (mmI

Fig. 1: A) Time-dependent changes in the specific activity Sx of 22Na in the xylem exudate of barley roots during washing-out with inactive solution; B) Rate of the transport (R') of Na+ to the xylem exudate and C) Rate of the plasmalemma efflux "co of Na+ from the root cortex before and after an addition of K+ (arrow) in the presence of Na+. F.S. = efflux from the free space. Starting at t=O (sodium + 22Na)-loaded roots were washed-out with non-radioactive solution (methods). The efflux of 22Na from the cortex and from the severed xylem vessels and the xylem transport of Na+ were measured separately. From the 22Na and the Na+ concentration in the xylem exudate the specific activity Sx was determined and plotted in A). Using Sx the xylem and cortical 22Na effluxes were converted to Na+ effluxes and plotted in B) and C). Conditions: 1 mM NaCI (+22Na during loading), 3 mM CaCI 2; pH 8, adjusted by titration with Ca(OH)2; aeration: 80 0J0 N 2 + 20 0J0 02; T = 22-23 0c. At t=360 min 0.2 mM KCI was added in the presence of Na+ (open arrows). 8. K+- dependent or H+-dependent Na+extrusion in re-elution experiments

As described previously (JESCHKE, 1973, 1977b), the efflux of 22Na from barley roots reaches a quasi-steady state about 4 h after the start of the elution. The cytoplasmic specific

Z. Pflanzenphysiol. Bd. 98. S. 155-175. 1980.

160

WOLF DIETER JESCHKE

actiVity Sc and hence the cortical efflux (IP* out(S) = "co' sc) or the efflux through the xylem vessels (IP"out(X) = R' . sc) then remain nearly constant (Fig. 1). Sc is then governed mainly by the vacuolar specific activity Sv (equation (8) in JESCHKE, 1977 b). When a small concentration of K+ is then added to the wash-out solution in S (or in both chambers), the cortical 22Na efflux is rapidly and transiently increased (JESCHKE, 1973 and Figs. 1 and 3). From the peak in the efflux curve (Fig. 1) and the specific activity in the cytoplasm Sc the K+-stimulated sodium efflux was calculated. By subtracting the plasmalemma efflux "co before addition of K+, the K+-dependent sodium efflux "co(K+-dep) was obtained. Similarly a proton-dependent sodium efflux was obtained in experiments in which the pH was abruptly changed at the beginning of «re-elution» (pH-jump experiments), see Figs. 5 and 8.

moJO' OtHons

(m"')

A

8

C

(35)

17

",(K'·dop)

(K'·dop)

No'tronsport R' (eMlroll

36

cr (7)

12

0.16

51

,63

0.8

1.95

Oe

5()i~~F'O<-

105

o

(K·~mh,b ltd

79

96

80

Fig. 2: Effect of the accompanying anions on K+-dependent (net) sodium efflux "co(K+-dep) (A, B) and on the transport R' of sodium to the xylem exudate of barley roots (C, D). Experimental conditions as shown for each of the anions in Table 2. A) K+-dependent net sodium efflux [,amol g fr.wt.- 1h- 1] in presence of 0.2 mM K+; B) the same, but as related to the cytoplasmic sodium content; C) transport of sodium to the xylem exudate, the numbers denote the uninhibited control [,amol g-1 fr.wt. h- 1 ]; the inhibited transport in the presence of 0.2 mM K+ is seen from the hatched columns; D) effect of 0.2 mM K+ on sodium transport (1 mM Na+). 0.2 mM K+ was added as KCl in the experiments with N0 3- or Cl- anions and as K 2 SO. in the sulfate experiments.

*

Mean of 5 experiments; the mean of 29 replications was 9.6

±

0.6.

Results 1. K+-dependent net sodium extrusion measured by tracer re-elution measurements Although K+-induced net sodium extrusion can be observed by changes in the external Na+ concentration (JESCHKE and STELTER, 1973), the difficulty of accurately detecting rather small concentration changes limits this method to those cases in which larger amounts of Na+ are extruded. A more generally applicable method was Z. Pflanzenphysiol. Bd. 98. S. 155-175. 1980.

Involvement of protons in K+-Na+ selectivity

01('

.01m/'lI('

O/('

161

,01 m'
InS

.... o

~

~

pH 4

O.1-,--~-~-~--{

200

300

tOO

!fiX)

f-:'==:::::!:~"':~==..-1 200

300

tOO

!fiX)

time (mm)

Fig. 3: K+ induced changes in the plasmalemma efflux "'co of Na+ (left) and in the transport of Na+ to the xylem exudate (right) in barley roots at different pH. As in Fig. 1 22Na fluxes were converted to Na+ fluxes. Conditions: pH 8 or 4, adjusted by titration with Ca(OH)2 or HCI; other conditions as in Fig. 1. developed by a modification of compartmental analysis (PITMAN, 1971; JESCHKE, 1972, 1977 b) in which the efflux of tracer, e.g. 22Na across the root cortex iP'~out(S) and its exudation across the xylem vessels iP" out(X) is measured, After an initial washing-out of the free space (see F.S. in Fig. 1 C) iP"out(S) is governed by the plasmalemma efflux 0 co and by the specific activity in the cytoplasm or the symplasm Sc:

(1)

Similarly, iP"out(X) is given by the net transport of Na+ to the xylem exudate R' and the specific activity in the xylem sap Sx:

<1>* out(X) = R" sX'

(2)

Hence Sx can be determined independently of the flux analysis if both the transport ofNa+ (R') and of2 2Na (iP*out(X)) are measured, see methods, No.7. Fig. 1 A shows the changes in Sx during an efflux experiment. After a short time-Iag1) Sx clearly reflected the change with time that can be expected in the 1) Since transport in the xylem vessels occurs at a definite velocity, any signal, like a decrease in the specific activity, reaches chambers X only after a time-lag that depends on the lenght of path between chamber Sand X. When Sx was used as a measure of sc' the time-lag was considered. Z. Pflanzenphysiol. Bd. 98. S. 155-175. 1980.

162

WOLF

DIETER JESCHKE

cytoplasmic specific activity Se during washing-out of a tissue (PITMAN, 1971; JESCHKE, 1977 b): an initial fast decrease (0-200 min in Fig. 1 A) - due to exchange across the plasmalemma - and a quasisteady slow decrease (200-360 min in Fig. 1 A) during which Se is determined by the vacuolar specific activity according to se(steady)

=Sv'

"ve

"co

+ R' + "cv

(3)

Thus Sx can be equated to Sc as would be expected, if Na+ is transported symplastically across the roots to the xylem vessels. Using the magnitude of sc(sx) at each timet) and the equations (1) and (2), the 22Na tracer effluxes
2. Effect of anions on Na+ fluxes and K+-dependent Na+-extrusion Until now K+-Na+ exchange in barley roots has been measured only using balanced solutions containing nitrate, phosphate, sulphate and chloride anions (e.g. JESCHKE, 1973, 1977b, c; JESCHKE and STELTER, 1973). On the other hand, K+ stimulated the 2) Steady state conditions and constant fluxes throughout the experiment are a prerequisite for flux analysis (PITMAN, 1971; JESCHKE, 1977 b). The steady fluxes seen in Fig. 1 therefore justify the application of this analysis. Z. Pflanzenphysiol. Bd. 98. S. 155-175. 1980.

Involvement of protons in K+-Na+ selectivity

163

22Na efflux in isolated root tips only when 504-- was the accompanying anion (RATNER and JACOBY, 1976). NO a- and 504 -- are known to induce OH- or H t excretion, respectively (JACKSON and ADAMS, 1963; 5MITH and RAVEN, 1976) and may thus affect other reactions involving proton fluxes. It was necessary, therefore, to study the effect of anions before investigating the influence of protons. In these experiments, nitrate and sulphate were used in combination with phosphate as a buffer in order to avoid unintentional changes in pH. With nitrate as the major counter-ion, high flux and transport rates of Na+ were found. CI- was intermediate while 504-- permitted only rather low rates (Table 2). Also the cytoplasmic Na+ content was somewhat smaller in presence of 504 -- than NO a-. In all cases K+ induced a net extrusion of cytoplasmic Na+ [0 co (K+-dep)) but the magnitude of the extrusion varied widely being smallest with 504 -- as the anion (Fig. 2 A). This was unexpected since cation exchange might be assumed to be independent of the accompanying anions (5LAYMAN and 5LAYMAN, 1968). However, since the cytoplasmic Na+ concentration is one of the factors limiting the rate of K+-Na+ exchange (JESCHKE, 1977 c), the smaller cytoplasmic Na+ content Qc (or hence Na+ concentration) in the presence of 504 -- (Table 2) can, at the least part, account for the smaller rate of Na+ extrusion. The anion dependency of Na+ extrusion was thus greatly reduced but not eliminated when 0 co (K+-dep) was related to Qc (Fig. 2 B). Furthermore, Na+ transport was strongly and selectively inhibited by K+ in the presence of each of the anions (Fig. 2 D). Clearly these results contrast to those of RATNER and JACOBY (1976) with excised root tips. This can not be attributed to the use of different tissues, however, since net Na+ extrusion in the presence of NO a- can be shown also in meristematic root tissues (JESCHKE, 1979 a).

3. Effect of protons on K+-Na+ exchange

If protons were directly involved in K+-Na+ exchange, the external pH might specifically affect this reaction, i.e. if Na+ efflux were mediated by Na+-H+ antiport, low pH could be expected to stimulate K+-Na+ exchange. In the studies involving changes in pH, only chloride was used as counter-anion in order to prevent uncontrolled OH- or H+ fluxes due to the presence of NO a- or 504--. The effect of external pH is seen in Tables 3 and 4. The cytoplasmic Na+ content, the Na+ fluxes, and particularly the transport of Na+ (R') responded to changes in pH (Table 3). All fluxes were severely decreased at pH 4 compared to 5.8 or 8. The vacuolar Na+ content was much less affected (Table 3), a similar observation was made on the effect of sulphate (Table 2). Potassium induced transient net Na+ extrusion and inhibited sodium transport at all 3 pH values, compare Fig. 3. The K+-dependent net Na+ efflux was apparently strongly decreased at pH 4 (Table 4, cpo Fig. 3). Yet if the cytoplasmic Na+ content is taken into account, then the difference between pH 8 and 4 is transposed (Table 4): Z. Pflanzenphysiol. Bd. 98. S. 155-175. 1980.

CaCl 2 (3)

chloride (7) 1.6±0.2

1.3 ±0.2

1.0 ±0.3

0.81 ±0.03

1.1 ±0.05

0 cv ,0 vC

72±3 81 ±3

104 ±0.1

69±5

74±3

Qv

0.14±0.04

0.16±0.02

2.5 ±0.14

R'

,umol

2.6±0.2

1.4±0.2

2.1 ±Oo4

3.9±0.2

Qe

g-1fr.wt.

1) Sodium fluxes were determined as described in the methods. Throughout the measurements the chamber X (compare insert in Fig. 1) contained only the basal medium, while chamber S contained in addition sodium as the salt indicated and 22sodium during the loading period, compare also table 1; the pH was 5.8; data ± standard error of the mean.

NaCI (1) 3.0±0.3

1.4±0.25

Na2HP04 (1)

1.6±004

CaS04 (0.5)

sulfate (0.5) + phosphate (1)

1.5±Oo4

1.7±004

Na 2HP0 4 (1)

CaS04 (3) MgS0 4 (0.5)

sulfate (3.5)

2.2±0.1

4.7 ±0.2

NaH2P 04! Na 2HP0 4 (1)

Ca(NO a)2 (3) MgS0 4 (0.5)

nitrate (6)

!'J eo

,umol !'Joe

addition in chamber S (mM)

major anions (mM)

basal medium1 ) (mM)

conditions, experimental solutions

g-1fr.wt.h- 1

Table 2: Effect of the accompanying anion on sodium fluxes and transport and on the cytoplasmic and vacuolar sodium content of barley roots 1 ).

4

4

5

29

number of replications

...... C1'

'"

(') '" ~

';;;<

""!

,.'"

t::l ;;

."

,~ ...

-l>-

Involvement of protons in K+-Na+ selectivity

165

Table 3: Effect of the pH on the steady state fluxes and cytoplasmic or vacuolar content of sodium in barley roots. ,amol g-l fr.wt.h- 1

lnditions Hon 11M)

Na+ (mM) pH 8 5.8 4

1- (7)

,amol g-l fr.wt. f2J cv =

0i vc

"'oc

"'co

3.6±0.2 3.0±0.3 0.9±0.1

1.510.1 1.2 ± 0.1 1.6±0.2 1.3±0.2 0.6 ± 0.05 0.5 ± 0.06

R'

Qv

2.2 ±0.2 1.4 ±0.01 0.27±0.05

95±3 81 ±3 54±4

number of replications

Qc 3.2±0.2 2.6±0.2 0.6±0.05

8 4 5

Sodium fluxes were determined as described in the methods. Conditions: The solution in chamber S (insert in Fig. 1) contained 1 mM NaCl + 3 mM CaCI 2 + 22Na during the loading periods, the pH was adjusted to the given values by titration; chamber X contained the basal medium with 3 mM CaCI 2 only; T = 24°Cj data ± standard error of the mean. Table 4: Potassium- or proton-dependent sodium efflux values. conditions: solution in chamber S NaCI CaCI 2 pH mM mM

additions during re-elution KCI pH mM

barley roots at different pH

"'co(K+-dep) 0 co (H+-dep) ,amol g- l fr.wt.h- 1

1 1 1

3 3 3

8 5.8 4

0.2 0.2 0.2

8 8 8

5.4±0.9 5.2±0.8 1.6±0.2

1

3 3

8 8

0.2

4 4

5.7±0.9

1

In

2.6±0.6

"'co(K+-dep) Qc

number of replications

1.6 2.0 2.5

8 4 5

(2.3)

6 4

Conditions as in Table 3, except 0.2 mM K+ was added to the roots orland the pH was changed from 8 to 4 after the roots had reached the quasi-steady state of efflux (at t = 360 min, d. Fig. 1); data ± standard error of the mean.

on this basis the K+-dependent Na+ efflux is enhanced at pH 4 and diminished at pH 8. Although not conclusive, the relative promotion of K+-Na+ exchange at acid pH could point to an involvement of protons in the process. A similar conclusion is also suggested by the low cytoplasmic Na+ content, Qc, at pH 4 (Table 3). If Na+ ions were extruded by H+-Na+ antiport, low pH would tend to decrease Qc. However, other explanations such as competition between Na+ and H+ for cytoplasmic binding sites cannot be ruled out. If the Low cytoplasmic Na+ content Qc at pH 4 were due to H+-Na+ antiport, the cytoplasmic H+ content should be high under these conditions (in the absence of K+). Consequently, potassium influx during re-elution should be high, since it would serve to extrude sodium as well as protons from the cytoplasm. The stoichiometry K+ Z. Pflanzenphysiol. Bd. 98. S. 155-175. 1980.

166

WOLF DIETER JESCHKE

1mM No·, anion C/-

'I

~

10

~l.;

'I""'

...g

C),

'0

•lc

"0 E

::l..

5

pH.

pH 8

~ ,0.2 mM K'/m 5)

~ ,O.2mM K'(in 5)

~""'"

10

5

No' efflux

No+ efflux

J~

0 300 .00

••• I

__

r-'-

K'fronsporf

500 600

.~.r_f

_rr

r

K+ transport

300 .00 500 600

time [min]

Fig. 4: Rates of net K+ uptake in and of K+ transport across barley roots during «re-elution» experiments at pH 4 and 8 as a function of time. For comparison the efflux 0 eo of sodium from the root cortex is included. Conditions and experiments as in Fig. 3. At t=360 min 0.2 mM KCl was added (open arrow). Net K+ uptake therefore starts at this time, K+ transport starts to rise after a long time-lag during which the roots equilibrate with KCI.

influx/Na+ efflux, therefore, should be considerably a:bove 1 at pH 4 but much lower at pH 8. This is clearly borne out by the results of Fig. 4, showing K+/Na' stoichiometries around 4 at pH 4 and 1.6 at pH 8. 4. Proton- and (proton

+ potassium)-stimulated Na+ efflux

Stronger evidence for Na+-H+ antiport would be obtained, if protons stimulated the efflux of 22Na. Roots were loaded with Na+(22Na) at pH 8 and then subjected to a jump in pH from 8 to 4 either by changing the buffer or in unbuffered solutions by titrating to the new, desired pH. In both cases acidification induced an increase in the cortical 22Na efflux (Fig. 5 and 7 A). But unlike the transient changes produced by K+ (Figs. 1 and 3) protons alone induced a prolonged and sluggish increase in Na+ efflux. This was not followed by a marked decrease in xylem transport (Fig. 5 and 7 A) as was found with K+ (Fig. 1 and 3). A similarly slow proton-stimulated efflux has been found in Streptococcus (HAROLD and PAPINEAU, 1972). The long duration of the increase suggests that the tonoplast as well a plasmalemma effluxes are affected. Thus it may be calculated from the area under the curve in Fig. 5 C that more than 8 ,umol Na+ g-lfr.wt. were lost from the roots after acidification and this exceeds Qe(Na) (3.3 ,umol g-lfr.wt. in this experiment, cpo Table 3, pH 8). An increase in vacuolar efflux 0 ve also follows from the slow Z. Pjlanzenphysiol. Bd. 98. S. 155-175. 1980.

Involvement of protons in K+-Na+ selectivity

167

I mM No+, onion C/-

7

.E!!.!..- _-,-p_H_'_ _

6x10' 4

A

2 IxIO'

c

(pH')

~

~

3~'"

R'

\ I Isc===+x I

1

t

L

8

8.l... .g -0

2§~:[;~E+O.c::

L.... '"

~ ;-

O+-_~_~_~ "r-~-~-~-~_-"-oi~l~ 200

300

400

500

200

300

400 500 fimefminl

Fig. 5: Proton-induced changes (A) in the specific activity Sx of 22Na in the xylem exudate, (B) in the efflux (transport) of sodium R' to the xylem exudate and (C) in the plasmalemma efflux "co from the root cortex in barley roots. Note the logarithmic scale for Sx in A. Procedure and experimental conditions as in Fig. 1 or 3, pH 8 except 0.1 mM H+ was introduced at t=360 min: i.e. the pH was changed from 8 to 4 (black arrows) in the chamber S.

increase in the cytoplasmic specific actiVIty Sc that was monitored by the specific activity in the xylem sap. sx, Fig. 5 A. The magnitude of "YC can be estimated using equation (3) since all other variables apart from the tonoplast influx "Cy are known 1 ). Assuming "Cy to be unchanged, "YC would be required to increase from 1.2 to 3 pmol g- l fr.wt. within 2 hours. Alternatively the increase in Sc could be accounted for by a decrease in the tonoplast influx "Cy; both of these changes would have the same over-all effect, i.e. sodium would tend to leak out at the acid pH, cpo JACOBSON et al. (1960). In any case the changes in "co preceeded those at the tonoplast as can be judged from the fast increase in "co and the fairly slow one in Sx (Fig. 5 C and A). This shows an independent stimulation of "co by protons. From this a proton-dependent Na+ efflux of 2.6 pmol g- l fr.wt. h- 1 was estimated, which was lower than the K+-dependent one induced by 0.2 mM K+ (5.4 pmol g- lfr.wt. h- 1)2). The results suggest the operation of a Na+-H+ antiport which is less efficient though than the full system mediating K+-Na+ exchange. 1) Sy can be obtained in several ways (JESCHKE, 1977 b), during steady state efflu,x it is obtained from the activity in the tissue and the vacuolar sodium content; Sc (or sx), "co and R' have been measured (Fig. 5). 2) At 0.1 mM K+, the concentration corresponding to pH 4, "co(K+-dep) will not be much smaller, since it is close to saturation already at 0.1 mM K+ (JESCHKE, 1977 b). Z. Pjlanzenphysiol. Bd. 98. S. 155-175. 1980.

168

WOLF DIETER JESCHKE

lmM Na+, anion C/o

...,0

~

9

E E

8

~ I

~

g II)

><<:

CIl-, .... 01 80 -~ ~~ +

-

i:l~

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~ ~

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5

(pHS)

~ ~

(pH 4

.K·)

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/

2

~.:: >< ..... 01 .2~

""

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4

o E 3 ";:, :J..

>< CIl

~

6

.... ° <.> ° ~CIl

~;:,

7

~

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+K'

pH 4

~~I

:}~

b3xl

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.go

....o:J.. E

'\.

~

....0

0

....

~

e 200

300

400

500

200

300

400

500 time [min]

Fig. 6: (Proton + potassium)-induced changes in the efflux "co of sodium from the plasmalemma of the cortical cells (5) and in the efflux (transport) R' to the xylem exudate (X) in barley roots. Procedure and experimental conditions as in Fig. 1 or 3, pH 8, except 0.1 mM H+ + 0.2 mM K+ were added at t=360 min: i.e. the pH was changed from 8 to 4 and 0.2 mM KCl was added (black and closed arrows) in the chamber 5.

If this is true, K+ given together with a pH-jump should result in a full stimulation of Na+ efflux. As is seen in Fig. 6, the combination of protons and K+ ions indeed induced such a strong, transient increase in net plasmalemma efflux of sodium and a selective decrease in the transport of Na+ to the xylem vessels. As would be expected, if K+-Na+ exchange were a function of H+-Na+ antiport, the (K+ and H+)-dependent Na+ efflux appeared to be somewhat higher than that with K+ alone (Table 4). If protons induce a slow increase in the tonoplast N a+ efflux, as shown above, the cytoplasmic Na+ content will tend to rise again after a pH-jump from pH 8 to 4. K· can be expected, therefore, to induce a second extrusion of these cytoplasmic sodium ions, if K+ is added subsequently to a jump in pH. K+ ions clearly induced a second increase in the cortical Na+ efflux, which was transient by contrast to the first, proton·induced one (Fig. 7 B). After the transient increase, the cortical sodium efflux returned to the levels seen before the addition of protons (Fig. 7 B) and the xylem transport of Na+ was inhibited to the levels obtained without a preceeding jump in pH (cp. Figs. 7 Band 1). This indicates that any pH-induced secondary changes such as the long-term increase in the vacuolar Na+ efflux (Fig. 7 A) were reversed after the addition of K+ (Fig. 7 B). Z. Pjlanzenphysiol. Bd. 98. S. 155-175. 1980.

Involvement of protons in K+-Na+ selectivity 200 Ix 10 5

2

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::l

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'-

I

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(pH 8)

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8

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-

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,O.2mM K'

600

400

,:;00

pH 4

~

4

~'-

400

pH8

pH8

-

t

(pH 8)

pH 4

1

-

(pH 4)

A

lxl0 J

169

'"

~::l ><

'"

E

..! i ~ -<: ...: .E ~ ><

::l

::::

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i

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200

400

200

400

600

time [min]

Fig. 7: Effect of A) a jump in pH from 8 to 4 or B) of a jump in pH from 8 to 4 followed by an addition of K' on the efflux of 22Na from the cortex (5) or to the xylem exudate (X) in 22Na(Na+)-loaded barley roots. Procedure and conditions as in Fig. 3, pH 8, except the pH was changed from 8 to 4 in A and B (black arrows) and subsequently 0.2 mM KCl was added in B (open arrows).

Discussion 1. Effect of anions on the steady state sodium fluxes

Counter-anions are known to affect uptake of cations and in particular their transport across the roots to the xylem vessels. Anion effects in the short-term cation influx of low-salt roots (EpSTEIN et aI., 1963) and of salt-pretreated roots (COOIL, 1974; JOHANSEN and LONERAGAN, 1975 a) depend on the concentration range and only small changes are seen at concentrations below 1 mM (<S04-- has recently been found in cucumber (COOIL, 1974) and similarly in barley roots (JoHANSEN and LONERAGAN, 1975 b). It was not known, however, which particular membrane or flux was responsible for these changes. Our data (Table 2) show the cation (Na+) influx across the tonoplast 0 cv and the vacuolar Na+ content to be rather insensitive to counter-anions, while other fluxes Z. Pjlanzenphysiol. Bd. 98. S. 155-175. 1980.

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WOLF DIETER JESCHKE

are strongly affected. Similarly, Na+ accumulation (in vacuoles) from sulphate-containing solutions was completed almost as fast (within about 15 h, data not shown) as from such with nitrate (JESCHKE, 1972). With sulphate as the counter-anion, cation uptake is accompanied by proton extrusion (JACKSON and ADAMS, 1963), organic acids are synthesised within the cytoplasm (HIATT, 1967), and their anions are assumed to accompany cation uptake across the tonoplast. The small response of the tonoplast Na+ fluxes to the counter-anion species and the unaltered rate of Na+ accumulation in the presence of sulphate appears to confirm the high efficiency of this intracellular anion generation. On the other hand, the xylem transport of sodium R' responded severely to anions and was decreased in the order NO a- > Cl- ~ S04-- (Table 2). Since the cytoplasmic Na+ content was relatively stable (Table 2), the depression of R' must be due to decreased loading of the xylem vessels with Na\ demonstrating that delivery of cations to the xylem sap requires a suitable anion. In theory Na+-H+ antiport across the symplasm-xylem boundary (HANSON, 1978) might be used to establish and maintain electroneutrality during secretion of cations to the xylem sap. This is not sufficient, however, to support xylem transport and volume flow. In agreement with the scheme of HANSON (1978) transport to the xylem vessels requires cation and anion effluxes from the symplasm, both of which may be powered by a proton pump. Sulphate, then appears only modestly suited for efflux across the symplasm-xylem boundary. 2. pH and steady state sodium fluxes

Data on the effect of alkaline pH on cation uptake are rather conflicting and complicated by possible interactions with CO 2 and HCO a-. A stimulation of cation uptake in beet (HURD and SUTCLIFFE, 1957) was thus attributed to an increase in HCO a- concentration (HURD, 1958) but might equally be ascribed to increased K+/H+ exchange at high pH (POOLE, 1976). In barley roots FALADE (1972) found no effect or an inhibitory one of pH 8 on K+ uptake in the absence of CO 2 while NG and POEL (1978) reported a stimulation by pH 8, more or less independent of CO 2 , In our experiments, the plasmalemma influx, the xylem transport, and the cytoplasmic content of sodium were increased at pH 8 compared to 5.8 (Table 3). As the vessels were aerated with 80 0J0 N 2 plus 20 0J0 O 2 and external CO 2 was thereby excluded and respiratory CO 2 largely removed, the increase appears to be due to the pH itself and not to HCO a-. Since proton extrusion will be favoured at high pH (POOLE, 1976), and Na+ influx can balance H+ extrusion in the absence of K+ (PITMAN, 1970; MARRE, 1977), improved proton extrusion may be the reason for the increased Na+ influx at pH 8. With respect to K+-Na+ selectivity and the involvement of protons, however, the effect of pH on K+ and Na+ fluxes in the presence of both ions should provide useful information and warrants further measurements. In Neurospora Na+ and K+ fluxes were differently affected by the external pH (SLAYMAN and SLAYMAN, 1970). Z. Pjlanzenphysiol. Ed. 98. S. 155-175. 1980.

Involvement of protons in K+-Na+ selectivity

171

Low pH values decrease the uptake of cations including Na+ and K+ and may even induce K+ leakage when Ca++ is absent (JACOBSON et al., 1960). Although 3 mM Ca++ was present in our experiments, pH 4 severely inhibited all fluxes and decreased in particular the cytoplasmic sodium content (Table 3). Superficially the effects of protons could seem related to those of sulphate (d. Tables 3 and 2). Both effects are quite distinct, however, in that the cytoplasmic sodium content is much more depressed at acid pH (Table 3) than in the presence of 504-- (Table 2). The decrease in the xylem transport at pH 4 therefore appears to originate from the low cytoplasmic sodium content only (Table 3) and not from an inhibition of xylem loading as with 504-- (see above). 3. Involvement of protons in K+-Na+ selectivity

A number of our results provide strong indications that protons mediate the highly efficient K+-Na+ exchange at the plasmalemma of barley root cells: a) although lower in absolute terms, the K+-dependent Na+ extrusion was favoured at low pH provided that it was related to the cytoplasmic Na+ content at each pH (Table 4); b) a sudden increase in the proton concentration to 0.1 mM (pH jump 8-+4) induced a proton-dependent Na+ efflux in sodium-loaded roots (Fig. 5); c) this effect (b) was greatly augmented when protons and K+ were added simultaneously (Fig. 6); and d) the low cytoplasmic Na+ content Qc at low pH (Table 3) could also imply proton-mediated Na+ efflux (Na+-H+ antiport). The latter argument is strengthened by the stoichiometric predominance of K+ influx compared to Na+ efflux at pH 4 (Fig. 4) suggesting that K+ serves to extrude protons as well as sodium ions at pH 4. Thus our results support the proposal of RATNER and JACOBY (1976) that protons mediate K+-Na+ selectivity, although there are significant differences between the two sets of data. The present results have been obtained with roots consisting predominantly of differentiated, vacuolated tissues. Proton fluxes across the plasmalemma therefore may have repercussions on fluxes across, or properties of, the tonoplast. In Fig. 8 a scheme is presented that may describe the involvement of protons in K+-Na+ selectivity (cp. HAROLD and PAPINEAU, 1972; RATNER and JACOBY, 1976; MARRE, 1977, 1979). The scheme includes the tonoplast in order to indicate possible interactions between the membranes. In this scheme Na+ efflux at the plasmalemma occurs in exchange for proton influx (H+-Na+ antiport, 3 in Fig. 8). Proton influx itself occurs down-hill (RAVEN and SMITH, 1977) but protons are re-extruded by a proton pump (1 in Fig. 8; MITCHELL, 1966) thereby regulating the cytoplasmic pH (RAVEN and SMITH, 1977) and maintaining the proton motive force across the plasmalemma. Proton extrusion is balanced electrically by K+ influx (2 in Fig. 8). The net effect is K+-Na+ exchange, while for protons the net flux is zero. Z. Pflanzenphysiol. Bd. 98. S. 155-175. 1980.

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WOLF DIETER JESCHKE

At the tonoplast only a proton pump (LIN et al., 1977) has yet been indicated (5 in Fig. 8). The affinities of the tonoplast for monovalent cation transport have been discussed in detail (JESCHKE, 1977 a, 1979 b). plasmalemma

outside (cell wall)

cytoplasm

vacuole

tonoplast

+

K'» No'

K', No'·

2

H' /:",'" H+

"

/1

: .. I

I

"\

" '

"

'.

Na+

H' An-

Fig. 8: Illustration of the possible relationship between the proton fluxes and K+-Na+ exchange at the plasmalemma and of a possible interaction with the tonoplast. For explanations, see text.

The site mediating K+ influx in exchange for protons at the plasmalemma (2 in Fig. 8) is suggested to have - at least in the low concentration range - the properties of the highly selective «system h (EpSTEIN, 1972), such as high affinity towards K+ and affinity sequence K+ ~ Na+. This follows from the properties of K+-Na+ exchange in barley roots (JESCHKE, 1977 b, c). Due to this selectivity sequence, proton-mediated K+-Na+ exchange can be «short-circuited» at higher Na+ concentrations. Na+ then can compete with K+ and partly balance for H+ extrusion, thus diminishing the over-all effectiveness of Na+-H+ antiport. This can be seen by the inhibition of K+-dependent Na+ efflux at higher external Na+ concentration (JESCHKE, 1977 c). The sequence K+ ~ Na+ (Fig. 8) is indicated also by the lower efficiency of Na+ in supporting H+ efflux (MARRE, 1977) particularly when the latter was stimulated by fusicoccin. Usually the concentrations of K+ and Na+ used to induce proton extrusion (JACKSON and ADAMS, 1963; PITMAN, 1970; MARRE, 1977) were higher than those corresponding to «system h. Moreover, K+ at concentrations below 1 mM was rather inefficient in supporting proton efflux (PITMAN, 1970). That K+ can balance proton extrusion from the cytoplasm with high affinity, i. e. at low concentrations, follows indirectly from the reversal of proton-induced changes by K+ (see Fig. 7 B and next Section). The scheme of Fig. 8 readily allows for a variable stoichiometry between K+ influx and Na+ efflux (JESCHKE, 1977 b) in that extruded protons can either re-enter the Z. Pflanzenphysiol. Bd. 98. S. 155-175. 1980.

Involvement of protons in K+-Na+ selectivity

173

cytoplasm in exchange for Na+ or by cotransport (symport) with anions (4 in Fig. 8; SMITH, 1970; SLAYMAN, 1974). Evidence for the involvement of proton fluxes in K+-Na+ exchange in barley comparable to that in bacteria (HAROLD and PAPINEAU, 1972) also has been obtained from the effect of inhibitors (RATNER and JACOBY, 1976). K+-dependent Na+ extrusion was strongly inhibited by CCCP and DCCD (JESCHKE, 1974; RATNER and JACOBY, 1976). The effect of DCCD, which inhibits the proton-ATPase of bacteria (HAROLD et al., 1969), suggests the involvement of a similar ATPase in K+-Na+ exchange of roots while the effect of the uncoupler CCCP indicates respiratory ATP to provide the energy (JESCHKE, 1974; RATNER and JACOBY, 1976). The plasmamembrane ATPase of plant roots has been suggested to act as a proton ATPase (HODGES, 1973) and it is sensitive to DCCD (LEONARD and HODGES, 1973). RATNER and JACOBY (1976) have also interpreted their observation that Na+ efflux was stimulated by K+ only in the presence of S04-- (see also POOLE, 1978), i. e. when there is net H+ extrusion in exchange for K+ (JACKSON and ADAMS, 1963) as avidence for proton dependency. However, this argument does not hold for differentiated root tissues since K+-Na+ exchange occurs in the presence of all anions (Fig. 2). Moreover, at a probable internal pH around 7 (SMITH and RAVEN, 1976) and a membrane potential of -90 mV (PITMAN et al., 1975) the proton motive force Ap is directed inwardly across the plasmalemma at external pH values around and below neutrality. Na+-H+ antiport therefore does not require net H+ extrusion for generating this force. As stated above, net proton fluxes would be zero during K+-Na+ exchange (Fig. 8 or HAROLD and PAPINEAU, 1972). In this connexion it is noteworthy that K+-Na+ exchange is still operative at pH 8. Since Ap may be expected to become inverted at more alkaline pH values (depending on the internal pH and the membrane potential A'lf), K+-Na+ exchange should decline at higher pH as was indeed observed, see the values of 0 co (K+-dep)/Qc in Table 4. The involvement of proton fluxes in K+-Na+ selectivity via H+-Na+ antiport is further supported by the recent finding (MARRE, 1979) that the fungal toxin fusicoccin can increase Na+ efflux from barley roots. This effect was attributed to proton fluxes (MARRE, 1979) since fusicoccin also stimulates the proton pump (MARRE, 1977). 4. The essential role of potassium ions

If Na+ efflux is coupled directly to H+ influx (Fig. 8), it is difficult to explain why acidification is only about half as effective in inducing Na+ efflux as is an addition of K+ (Table 4). According to the model, however, protons cannot be re-extruded when net Na+-H+ exchange occurs with externally supplied protons only. Na+ cannot efficiently substitute for K+ in balancing H+ extrusion since it would only short-circuit Na+-H+ exchange. Thus the cytoplasm will slowly be acidified and intracellular functions such as transport across the tonoplast could be affected by a decrease in pH. Thus the proton pump to the vacuole (LIN et aL, 1977; 5 in Fig. 8)

z. Pflanzenphysiol. Bd. 98. S. 155-175. 1980.

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WOLF DIETER JESCHKE

might be accelerated and this must be balanced by increased anion influx or cation efflux. In sodium-loaded roots, an increased sodium efflux may be expected. Alternatively membranes may become leaky at low pH (JACOBSON et al., 1960). In both cases the H+-dependent Na+ efflux from the cytoplasm will be followed by increased sodium efflux from the vacuole (Figs. 5 and 7 A). According to the model (Fig. 8) one function of K+ is removal of protons and thus regulation of the cytoplasmic pH (RAVEN and SMITH, 1977). The proton-induced changes should be reversed, therefore, if potassium is added after a pH-jump. As can be seen by a comparison of Fig. 7 A (acidification only) and B (acidification followed by addition of K+), K+ clearly can reverse the effects of protons; after a transient stimulation the sodium efflux from the cortex was decreased and xylem transport was inhibited to levels obtained without a preceeding acidification (Fig. 1). Taken together, the proton-induced increase in tonoplast efflux (Fig. 5 and 7 A) and its reversal by added K+ lend further support to the role of protons in mediating K+-Na+ selectivity as in the scheme of Fig. 8. Moreover they point to the essential role of K+ in stabilizing the cytoplasmic pH, a function Na+ ions cannot perform, when a H+-Na+ antiport is present. Admittedly this interpretation requires direct measurements of the proton fluxes. Preliminary results shown that net proton influxes in the presence of Na+ can be inhibited without delay, when K+ is added in the presence of Na+ (JESCHKE, unpublished). Acknowledgements This investigation was supported by a grant of the Deutsche Forschungsgemeinschaft. The skillful and untiring assistance of Mrs. HEDWIG BLUMEL in the experiments and drawing of the graphs is gratefully acknowledged. Thanks are extended to Mr. RUDOLF BEHL for help in developing the programs for flux evalution, to Drs. GARETH WYN JONES and JOHN TENHUNEN for reading the manuscript and improving the language, and to Mr. J. OEHLING and H. KOBOLD for constructing the experimental vessels.

References COOIL, B. J.: Plant Physioi. 53, 158-163 (1974). EpSTEIN, E.: Mineral nutrition of plants: principles and perspectives. Wiley and Sons, New York, London, Sydney, Toronto, 1972. EpSTEIN, E., D. W. RAINS, and O. E. ELZAM: Proc. nat. Acad. Sci. U.S. 49, 684-692 (1963). FALADE, J. A.: Canad. J. Bot. 50,1567-1579 (1972). HANSON, J. B.: Slant Physioi. 62, 402-404 (1978). HAROLD, F. M. and D. PAPINEAU: J. Membrane BioI. 8, 45-62 (1972). HAROLD, F. M., J. R. BAARDA, C. BARON, and A. ABRAMS: J. BioI. Chern. 244, 2261-2268 (1969). HIATT, A. J.: Plant Physioi. 56, 233-245 (1967). HODGES, T. K.: Advan. Agron. 25, 163-207 (1973). HURD, R. G.: J. expo Bot. 9,159-174 (1958). HURD, R. G. and J. F. SUTCLIFFE: Nature (Lond.) 180,233-235 (1957).

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JACKSON, P. C. and H. R. ADAMS: J. gen. Physiol. 46, 369-386 (1963). JACOBSON, 1., D. P. MOORE, and R. 1. HANNAPEL: Plant Physiol. 35, 352-358 (1960). JOHANSEN, C. and J. F. LONERAGAN: Aust. J. Plant Physiol. 2, 75-83 (1975 a). - - Z. Pflanzenphysiol. 75, 415-421 (1975 b). JESCHKE, W. D.: Planta 106, 73-90 (1972). In: ANDERSON, W. P. (Ed.): Ion transport In plants, 285-296, Academic Press, London, New York, 1973. - In: ZIMMERMANN, U. and J. DAINTY (Eds.): Membrane Transport in Plants, 397-405, Springer, Berlin, Heidelberg, New York, 1974. - In: MARRE, E. and O. CIFFERI (Eds.): Regulation of cell membrane activities in plants, 63-78. Elsevier, North Holland, Amsterdam, 1977 a. - Effects of K+, Rb+, Cs+, and Li+ on the Na+ fluxes. Z. Pflanzenphysiol. 84, 247-264 (1977b). - ]. expo Bot. 28, 1289-1305 (1977 c). - Z. Pflanzenphysiol., 94, 325-3'30 (1979 a). - In: LAIDMAN, D. 1. and R. G. WYN JONES (Eds.): Recent advances in the biochemistry of cereals, 37-61. Academic Press, London, New York, San Francisco (1979 b). JESCHKE, W. D. and W. STELTER: Planta 114, 251-258 (1973). LEONARD, R. T. and T. K. HODGES: Plant Physiol. 52, 6-12 (1973). LIN, W., G. J. WAGNER, H. W. SIEGELMANN, and G. HIND: B.B.A. 465,110-117 (1977). LtiTTGE, U. and G. G. LATIES: Plant Physiol. 41,1531-1539 (1966). MARRE, E.: In: MARRE, E. and O. CIFFERI (Eds.): Regulation of cell membrane activities in plants, 185-202. Elsevier, North Holland, Amsterdam, 1977. - In: LAIDMAN, D. 1. and R. G. WYN JONES (Eds.): Recent advances in the biochemistry of cereals, 3-25, Academic Press, London, New York, San Francisco, 1979. MITCHELL, P.: BioI. Rev. 41, 445-502 (1966). NG, S-Y. and 1. W. POEL: Ann. Bot. 42, 411-418 (1978). PITMAN, M. G.: Plant Physiol. 45,787-790 (1970). - Aust. J. bioI. Sci. 24, 407-421 (1971). PITMAN, M. G. and H. D. W. SADDLER; Proc. nat. Acad. Sci. (Wash.) 57,44-49 (1967). PITMAN, M. G., N. SCHAEFER, and R. A. WILDES: Planta 126, 61-73 (1975). POOLE, R. ].: In: LUTTGE, U. and M. G. PITMAN (Eds.): Encyclopedia of plant physiology, New series, Vol. 2 A, 229-250, Springer Verlag, Berlin, Heidelberg, New York, 1976. - Ann. Rev. Plant Physiol. 29, 437-460 (1978). RAINS, D. W. and E. EpSTEIN: Plant Physiol. 42, 319-323 (1967). RATNER, A. and B. JAOOBY; J. expo Bot. 100, 843-852 (1976). RAVEN, J. and F. A. SMITH: In: MARRE, E., and O. CIFFERI (Eds.): Regulation of cell membrane activities in plants, 25-40. Elsevier, North Holland, Amsterdam, 1977. SLAYMAN, C. 1.: In: ZIMMERMANN, U. and]. DAINTY (Eds.): Membrane Transport in Plants, 107-119. Springer, Berlin, Heidelberg, New York, 1974. SLAYMAN, C. 1. and C. W. SLAYMan: ]. gen. Physiol. 52, 424-443 (1968). - - ]. gen. Physiol. 55, 758-786 (1970). SMITH, F. A.: New Phytol. 71, 595-601 (1970). SMITH, F. A. and]. RAVEN: In: LUTTGE, U. and M. G. PITMAN (Eds.): Encyclopedia of Plant Physiology, New Series Vol. 2 A, 317-346. Springer, Berlin, Heidelberg, New York,1976. WAGNER, G., R. HARTMANN, and D. OESTERHELT: Eur.]. Biochem. 89, 169-179 (1978). W. D. JESCHKE, Botanisches Institut I, Universitat Wiirzburg, Mittlerer Dallenbergweg 64, D-8700 Wiirzburg.

Z. Pjlanzenphysiol. Bd. 98. S. 155-175. 1980.