Journal of Experimental Botany, Vol. 36, No. 162, pp. 63-78, January 1985

Potassium Transport in Enteromorpha intestinalis(L.) Link.

I. MEASUREMENT OF INTRACELLULAR K + , EXCHANGE FLUXES ANDTHERMODYNAMIC ANALYSIS

RAYMOND J. RITCHIE AND A. W. D. LARKUM

The School of Biological Sciences, The University of Sydney, Sydney, N.S.W. 2006, Australia

Received 27 March 1984

ABSTRACTPotassium transport has been studied in the marine euryhaline alga, Enteromorpha intestinalis culturedin seawater and in low-salinity medium (Artificial Cape Banks Spring Water, ACBSW; 25-5 mol m~3

Cl~, 20-4 mol m" 3 Na+ , 0-5 mol m~3 K+) . K+ fluxes were measured using *2K+ and 8 6 Rb + although8 6Rb+ does not act as an efficient K + analogue in this plant. 4 2K + experiments on seawater plantstypically exhibited a single protoplasmic exchange phase whereas 8 6 Rb + exhibited two exchangephases. Compartmental analysis of 8 6Rb+ efflux experiments on seawater-grown Enteromorpha plantswere used to deduce the intracellular partition of K+ between the cytoplasm (279 ±38 mMolal) andvacuole (405 ±68 mMolal). The plasmalemma K+ flux in plants in seawater was greater in the lightthan in the dark (563±108 nmol m" 2 s"1 versus 389±66-7 nmol m~2 s"1). In low-salinity plants,separate cytoplasmic and vacuolar exchange phases were apparent. Analysis of *2K + effluxexperiments on low-salinity plants yielded a cytoplasmic K.+ of 222 ±38 mMolal and a vacuolar K + of82±11 mMolal. The plasmalemma and tonoplast flux was 23±4-5 nmol m " 2 s"1.

The Nernst equation showed that, although K+ was close to electrochemical equilibrium, activeaccumulation of K+ across the plasmalemma occurred in plants in seawater and ACBSW both in thelight and dark. K+ was also actively transported inwards across the tonoplast in low-salinity plants.The electrochemical potential for K+ across the plasmalemma ranged from 2-41 ±0-60 kJ mol"1 inplants grown in seawater in the light to 5-79 ±0-87 kJ m o P 1 for plants in ACBSW in the light.Although K.+ is close to electrochemical equilibrium, the flux of K+ in plants in both seawater andACBSW media is high, hence the power consumption of K + transport is high. The permeability of K +

(PKf) was significantly higher in the light than in the dark in plants in seawater (about 7-0 versus2-5 nm s"') but in plants in low-salinity (ACBSW) medium the permeability was independent of light(about 12 nm s"1). The energy requirements of active K+ transport by ATP-dependent pumps isdiscussed.

Key words; Enteromorpha; Potassium transport; Ionic relations; Saltwater, Low salinity; Thermo-dynamics.

INTRODUCTIONThe present paper forms part of a study of the ionic relations of the multicellular euryhalinealga, Enteromorpha intestinalis (L.) Link. Aspects of the Cl" and Na+ ionic relations havebeen reported previously (Ritchie and Larkum, 1984a, b). This paper deals with themeasurement of intracellular potassium content, protoplasmic compartmentation andexchange fluxes of K+ for plants in both seawater (101 mol m~3 K+) and low-salinitymedium (0-5 mol irT3 K+), using 42K+ and 86Rb+.

64 Ritchie and Larkum—Potassium Transport in Enteromorpha

Knowledge of K + activity and fluxes in marine algal cells is largely based on giant algalcoenocytes (Gutknecht and Dainty, 1968; Hope and Walker, 1975; Raven, 1976) involvingmicrosurgical techniques which cannot be used in small-celled algae such as Enteromorphawhere intracellular contents and compartmentation must be deduced from compartmentalanalysis of 4 2 K + and 8 6 Rb + exchange kinetics (Atkins, 1969; Walker and Pitman, 1976).

The measurement of 4 2K + fluxes in Enteromorpha is simpler than 22 Na + fluxes becausethe intracellular K + compartment is relatively large and is not masked by the exchange ofcell wall bound 4 2 K + (Ritchie and Larkum, 1984&). Nevertheless, the cation exchange pro-perties of the cell wall of Enteromorpha (Ritchie and Larkum, 1982a, b) have to be taken intoaccount since the exchange kinetics of 4 2K+ would be very similar to 2 2Na+ in the cell wall.

In some biological systems, the alkali metal rubidium, behaves as an analogue for K + andhence 8 6 Rb + , with a half-life of 18-77 d has been used as a tracer of K+ . Unfortunately plantsusually discriminate against Rb + , e.g. Anabaena variabilis (Reed, Rowell, and Stewart, 1981),Nitellopsis obtusa (MacRobbie and Dainty, 1958), Porphyra purpurea (Reed and Collins,1981a) and Chaetomorpha darwinii and Ulva lactuca (West and Pitman, 1967a). Usually the8 6 Rb + flux is smaller than that obtained using 4 2 K + but the calculated intracellular K +

content, based on 8 6 Rb + labelling, is similar to that calculated using 4 2 K + (e.g. Anabaena,Reed et al., 1981; Porphyra, Reed and Collins, 1981a). 8 6 Rb + kinetic data have been usedto deduce the internal compartmentation of K + where 4 2 K + labelling experiments yieldedno such information (Ulva, West and Pitman, 1967a, b; Porphyra, Reed and Collins, 1981a).The kinetics of 4 2 K + and 8 6 Rb + are best compared in double labelling 4 2 K + / 8 6 Rb +

experiments.In the work reported here a thermodynamic analysis was made to determine if K + is out of

thermodynamic equilibrium under steady state conditions using the Nernst criterion. Theenergy required for K + transport was then calculated. Finally, the Goldman equation wasused to calculate the apparent permeability of K + across the plasmalemma (see, for example,Reed and Collins (1980) who worked on the red alga Porphyra). The results are discussed interms of the involvement of ATP-dependent mechanisms, as has been done in other algae(Hope, 1971; Hope and Walker, 1975; Raven, 1976).

T H E O R Y

Uptake flux experiments

Uptake of an isotopic tracer by a single intracellular phase follows an exponential saturationcurve. The uptake flux <f>0, is,

and for very short labelling times,

(1)

where, C * a is the total isotope taken up at isotopic equilibrium into compartment 1expressed in ct. min~1, C*A is the isotope taken up in time interval t into the cell (i.e. totaltissue uptake minus that into the free space) in ct. min ~ l , kl is the exchange constant (s ~s), so

is the specific activity of the bathing electrolyte (ct. min"1 mol"1).Provided the uptake times are short enough, e~b approximates l—kt, but such

experiments may be impractical (Cram, 1975). The equation for the simple exponentialsaturation curve can be rearranged to evaluate fc,,

)

Ritchie and Larkum—Potassium Transport in Enteromorpha 65

The above equations are strictly valid only if there is a single intracellular exchange phase(MacRobbie, 1971; Cram, 1975; Walker and Pitman, 1976). If a very short labelling time isused (i.e. where t -*• 0), the flux across the plasmalemma into the cytoplasm (<f>o c) of aeukaryotic plant approximates <f>oi in equation 2.

Uptake of isotope into the cytoplasm may be so rapid that the isotopic content of thecytoplasm soon reaches a quasi-steady state (Pallaghy and Scott, 1969; MacRobbie, 1971;Cram, 1975, Walker and Pitman, 1976). This was found to be the case in Enteromorpha plantsin low-salinity. In such cases longer term uptake experiments actually measure the uptakerate into the vacuole (the vacuolar flux <f>0 v and exchange constant fc2). If <f>o,c is largecompared to the tonoplast flux (f>cv and k2t is much less than unity,

<t>o,^dc;.jdtso. 0)

Double labelling experiments

Double labelling experiments were carried out to compare directly the 4 2 K + and 86Rb +

exchange kinetics on the same tissue sample in the course of the same efflux experiment. Eachcounting aliquot is counted twice about 2 d apart, where the intervening time (tj —12) isknown. Using a system of simultaneous equations the radioactive count of a countingsample can be separated into counts due to 4 2 K + and 8 6 Rb + respectively at an arbitrarytime of t = 0. The separate 4 2 K + and 8 6 Rb + counts are then corrected for decay during thecourse of the experiment.

The Nernst and Goldman equations

The application and limitations of the Nernst and Goldman equations have beendiscussed previously (Ritchie and Larkum, 1984a). The Nernst equation was used tocalculate the equilibrium potential for K + across the plasmalemma (EK^0) and between thevacuole and the outside (£K+,v,o) where the subscripts (c), (v) and (o) designate the cytoplasm,the vacuole and outside (bathing electrolyte) respectively. Enteromorpha is a small-celledeukaryotic organism and so the use of the Nernst criterion is not straightforward. Tonoplastelectrical potentials may be significant. It is necessary to deduce cytoplasmic and vacuolarK+ concentrations from compartmental analysis and then convert these concentrations toactivities using estimates of activity coefficients calculated from tables in Robinson andStokes (1968, Appendices 8.9 and 8.10). K+ activity in the bulk medium and in the cyto-plasm and vacuole were taken as equal to those of K G solutions of similar K +

concentration.The electrochemical potential for K + (AjIK+) was then calculated from the difference

between the K + equilibrium potential and the measured membrane potential (^1>o). K +

would be in electrochemical equilibrium if ApKf0 = 0 (Nicholls, 1982; Ritchie andLarkum, 1984a).

The use of the Goldman equations to calculate the apparent permeability of ions isoutlined in Nobel (1974) and Ritchie and Larkum (1984a). The Goldman flux equations onlyapply to two compartments separated by one membrane, thus the cytoplasmic concentrationof an ion ([CJiC]) is necessary to calculate a passive permeability based on a passive efflux outof a eukaryotic cell like Enteromorpha. No attempt has been made to calculate PK* acrossthe tonoplast.

M A T E R I A L S A N D M E T H O D SGeneralLaboratory cultures of Enteromorpha intestinalis (L.) Link were cultured and grown as describedpreviously (see Ritchie and Larkum, 1982a). The culture media used were enriched seawater (f/2; Stein,

66 Ritchie and Larkum—Potassium Transport in Enteromorpha

1973) or Artificial Cape Banks Spring Water (ACBSW) containing 25-5 mol m~3 Cl~, 20-4 mol m" 3

Na+ , 05 mol m " 3 K + (Ritchie and Larkum, 1982a). Artificial seawatcr based on AES (Stein, 1973) wasused in all sea water experiments to ensure that the seawater contained 101 mol m " 3 K + . Techniquesand general properties of Enteromorpha plants, based on CaCNO3)2 rinsing, have been publishedelsewhere (Ritchie and Larkum, 1984a, Table 1). All experiments were conducted at 20± 1 °C, pH 81(seawater) and pH 7-85 (ACBSW). The fresh weight and dry weight of tissue samples were determinedas described by Ritchie and Larkum (1982a). Intracellular K+ and flux rates were first calculated on adry or fresh weight basis then converted to mMolal and mol m~2 s~' respectively. All error-bars citedare means ±95% confidence limits with the number of replicates (n) or degrees of freedom (d.f.) inbrackets.

42KC1 and 86RbCl were purchased from the Australian Atomic Energy Commission, Lucas Heightsand from New England Nuclear respectively. The *2K + specific activities used were in excess of 30000Bq mol"1 . Samples were counted using either a Nuclear-Chicago gas flow counter or by Cerenkovradiation counting using a scintillation counter (Kobayashi and Maudsley, 1974).

Efflux experimentsComplete isotopic equilibration of K+ occurred in 12 h but 24 h loading times were used routinely.

The normal loading time was extended for 3 d for 8 6Rb+ efflux experiments because 8 6Rb+ equilibratedmore slowly than 4 2K + . In the 8 6Rb+ /*2K+ double labelling experiment the alga was first labelled inseawater containing 8 6Rb+ for 3 d, then 42K.+ was added to the labelling solution and the cells werelabelled in the presence of both isotopes for a further 24 h.

Labelled Enteromorpha plants were placed in a chamber and a peristaltic pump used to passunlabelled media past the labelled plants at 1-7 cm3 min~' (Ritchie and Larkum, 19826). The effluentwas collected using an aliquot collector. The 'light' treatment had been in the light for about 12 h beforethe commencement of the experiment. Similarly 'dark' efflux experiments were conducted on plantswhich had been in the dark for the previous 12 h. Both light and dark experiments were replicated.Efflux experiments were analysed using compartmental analysis as described by Atkins (1969),Pallaghy and Scott (1969) and Walker and Pitman (1976).

Uptake experimentsEfflux experiments on both seawater and low-salinity-grown plants showed that the 4 2 K + efflux

rates were very high and so short labelling times were necessary to measure uptake rates. Rinsingroutines to remove 42K.+ bound to the cell wall were not appropriate, so total 4 2 K + uptake (i.e. freespace + intracellular uptake) was measured as a function of time. The uptake rate was then calculatedby linear regression methods (Steel and Torrie, 1981). 4 2 K + uptake was measured on a fresh weightbasis (mol g"1 s"1) then converted to mol m~2 s"1 using data from Ritchie and Larkum (1984a).

RESULTS

Efflux rates and intracellular compartmentation

(A) Seawater plants

Figure 1 shows 4 2 K + efflux from seawater grown Enteromorpha plants in seawater in thelight. After an initial very short phase presumed to be from the 'free space' (apparent for only10-15 min) there was only a single exchange phase, as found in Ulva (West and Pitman,19676) and Porphyra (Reed and Collins, 1980, 1981a). A total of 23 light and 22 dark effluxexperiments exhibited a single intracellular exchange phase. Two manipulative experimentsexhibited two intracellular exchange phases and are dealt with separately.

Statistical analysis (ANOVA and F tests, P < 0-05 level; Steel and Torrie, 1981) of the4 2 K + fluxes showed that fluxes were about 50% higher in the light than in the dark (Table 1,part A). Similarly, ANOVA analysis showed that the data for total intracellular *2K+ couldbe pooled to give an overall mean based on 45 observations (Table 1, part A). Meanintracellular [K,+] were converted from a g dry weight (gDW) basis to mMolal (mol kg"1

H2O) units as described previously (Ritchie and Larkum, 1984a).

Ritchie and Larkum—Potassium Transport in Enteromorpha 674 2 K + exchange data on plants in seawater could not be used to deduce the intracellular

compartmentation of K+ (Walker and Pitman, 1976). The double labelling experiment inFig. 2 clearly shows that 86Rb + and *2K+ have different exchange kinetics, a result whichis very similar to that of Reed and Collins (1981a) working on Porphyra. 8 6 Rb + effluxexperiments always exhibited two intracellular phases (Table 1, part B). If it is assumed thatthe slow phase represents the vacuole and the fast phase, the cytoplasm, then the intracellular

' t 2 K E f f l u x cu r ve f o r S e a w a t e r P l a n t s in t h e L i g h t ( 3 )

JS.5

Single phase

L n E f l 1 =9 721

k, = 100173 mn

n = 5 9 , r = -0-999

,-1

0 10^ 20_^ 30 ALIQUOT N° 40 50 60

0 50 100 150 200 250 271TIME (minutes)

F I G . 1. * 2K + efflux kinetics by seawater grown Enteromorpha plants in the light. Rate of loss of 4 2 K + isplotted as Log. (counts remaining in tissue) versus time. After a very short phase of free space' exchange,

there was only one apparent intracellular exchange phase.

compartmentation of K + can be inferred from the 8 6Rb+ data as described by West andPitman (1967a, b) and Reed and Collins (1981a). Based on microscopic observations, thecytoplasm was estimated to occupy 25% of the protoplasmic volume. The tonoplast area hasbeen assumed to approximate the plasmalemma surface area as described previously for36C1~ transport in Enteromorpha (Ritchie and Larkum, 1984a). Table 1, Part B shows thatthe 8 6Rb+ data yields an apparent concentration of K + of 279 ± 38 mMolal in the cytoplasmand 405±68 mMolal in the vacuole. The total intracellular K + ([Kj+]), cytoplasmic K +

([Kc+]) and vacuolar K + ([Kv+]), based on 8 6Rb+ labelling were not significantly different

in the light and dark and so overall means based on 24 experiments (12 light and 12 dark)were calculated (Table 1, part B). The 8 6 Rb + plasmalemma fluxes in the light and the darkare significantly different from each other (t-test, P < 0-05, Steel and Torrie, 1981); the darkrate is about 60% of the light rate. When the 8 6 Rb + fluxes are compared to those obtained

68 Ritchie and Larkum— Potassium Transport in Enteromorpha

T A B L E 1. Intracellular \_K+~\ and exchange fluxes of Enteromorpha

(Based on Ritchie, 1982)

Part A. Values obtained using *2K +

(a) Low-salinity (ACBSW) mediumIntracellular[K,+] Total Cytoplasm"

mMolal

Effluxes

nmol g"1 DW s" :

nmol m" 2 s"1

(b) SeawaterIntracellular [K,+]

/jmol g"1 DWmMolal

Effluxes

122±9-5(36)* 57 + 9-3118± 12(50) 222±38

Plasmalemma (<f>cc)(Light and Dark)

1 44± 11(36)221 ± 5 4 0

Total

322+13-5(45)349 ±23-2(59)

Plasmalemma (<f>co)

Vacuole"

63 ±7-282±11

Tonoplast (̂ TiC)(Light and Dark)

4-5 ±0-85(36)23 ±4-5 0

Light Darkn m o l g - ' D W s " 1 103± 17-9(23) 71± 10-6(22)nmol m " 2 s " ' 563 ± 108 0 389 ± 66-70

Part B. Compartmentation based on 8 6 Rb + labelling

Intracellular

(j.mo\ g~'mMolal

Effluxes

nmol g~'nmol m~2

[K,+]

DW

D W s "

Total

345 ±45(24)371 ±51(38)

Plasmalemma

Light

1 84± 17(12)457 ±940

Cytoplasm"

64 ±8-2279 ±38

Dark

49± 17(12)267 ±88(0

(seawater only)

Vacuole"

283 ±44405 ±68

Tonoplast- (Light and Dark)

4-2 ±0-66(24)23±4-l

" The concentrations are calculated on the assumption that the vacuoleoccupies 75% of the protoplasmic volume.

* The values in parentheses refer to the number of replicates or (for mMolaland nmol m~2 s"1) to number of degrees of freedom.

c Signifies degrees of freedom in excess of 120.

using 4 2 K + (Table 1, part A), the 8 6Rb+ flux value across the plasmalemma in the light isnot significantly different from the 4 2K+ value. The 8 6 Rb + flux across the plasmalemmameasured in the dark is not only lower than the 8 6 Rb + flux in the light but also lower thanthe 4 2 K + flux rate in the dark. Evidently 8 6 Rb + is a good analogue for studying themovement of 4 2K + across the plasmalemma in the light but not in the dark. The 86Rb + fluxat the tonoplast was not affected by light and dark.

13

12

§•a 10

Ritchie and Larkum—Potassium Transport in Enteromorpha

4 2 K * / 8 6 R b * Double Label Efflux Exp. Seawater. Dark

8 6 R b * Slow phase

69

12

10

86Rb Fast phase:

L n E 0 1 = 1 0 04

k2 = - 0 0438 mm"

n « 9 . r = - 0 - 9 9 6

8 10 Aliquot N°

10 20 30 40 50 minutes

10 20 30 ALIQUOT N° 40

k 2 ="640 x 1 0 " 6 rnin"1

n=51,r =-0-994

42 K Single phase

LnE Q 1 =12-18

k^ = - 0 0146 mm

n = 60,r = - 0 9 9 7

-1

50 6050 100 150

TIME (minutes)200 250 271

FIG. 2, * 2K+ /* 6Rb+ double labelling efflux experiment on seawater on seawater grown Enteromorphaplants in the dark. The exchange kinetics of *2K+ exchange shows a single Lntracellular exchange phase(as in Fig. 1). The exchange kinetics of 8 6Rb+ is very different to that of K + . There are two intracellular

phases which can be resolved from one another using compartmental analysis.

(B) Experiments on plants in low-salinity (ACBSW) medium

A 4 2 K + efflux experiment on Enteromorpha plants grown and labelled in low-salinity(ACBSW) medium in the light is shown in Fig. 3; there are two intracellular exchange phasesand so [Kc

+] and [Kv+] could be deduced and separate plasmalemma and tonoplast fluxes

calculated. There was a total of 20 efflux experiments in the light and 18 in the dark.ANOVA methods showed that all the data on 4 2 K + labelling and fluxes could be pooled toyield means based on 36 replicates. 4 2 K + labelling and flux data are summarized in Table 1,part A.

A2K+ uptake experiments4 2 K + uptake experiments were performed to validate the steady state assumption. The

uptake rate of 4 2 K + by plants in seawater was too fast for the linear model to be adequateover the time-scale used. The linear model for uptake is only appropriate if — kt is muchless than one. In the seawater efflux experiments the average k is about —320 x 10~6 s~'(calculated from the mean efflux rate in Table 1, part A); thus in 50 min 1 — kt reached a valueof 0-96 or over 50% isotopic equilibration in less than 40 min. In contrast, the linear model for4 2 K + uptake by ACBSW plants yielded an estimate of the vacuolar flux very similar to theestimate from efflux experiments. Thus the linear model was quite adequate to measure the

70

13

V)

c

12

Ritchie and Larkum—Potassium Transport in Enteromorpha

4 2 K + 0 5 m M K + A C B S W , L igh t I t )

13

12

\ !Fast phase

LnE01=11 71

k1 =-00521 mn"1

n=12,r = -0 999

0 2 4 6 8 10 12 \k ALIQUOT N°

20 40 60 TIME(min)

Slow phase

i

k2 =-000531 diin"1

* . . n= 27, r =-0-999

10 20 ALIQUOT N° 30

50 100TIME minutes

150 200

FIG 3. *2K + efflux kinetics of low-salinity grown Enteromorpha plants in the light. There are twointracellular exchange phases resolvable by compartmental analysis.

TABLE 2. *2K+ uptake fluxes by EnteromorphaThe uptake fluxes shown in Table 2 were based on uptake rates on a fresh weight rather than dry weightbasis. The calculated uptake flux shown in Table 2 for seawater-grown plants is apparently theplasmalemma flux. In contrast, the uptake experiments on low-salinity plants measured the vacuolarflux rather than the plasmalemma flux.

Growth conditions Light/dark Uptake flux(nmol g"1 s" fresh wt.)

Uptake flux(nmol m~2 s

Seawater (20)plants

ACBSW (64)plants

LightDark

Light/dark

17 ± 3-69-8 ±3-41 2±O13

299 ±71173±6320 ±2-8

vacuolar flux of ACBSW plants but the cytoplasmic uptake phase could not be quantified(see Theory).

Table 2 shows that the 4 2 K + uptake fluxes of seawater plants calculated using equation 2are significantly higher in the light than in the dark, as found in the efflux experiments.Unfortunately, the uptake fluxes were significantly below the efflux values. This discrepancywill be discussed later. A linear model for 4 2 K + uptake could be used to determine the

Ritchie and Larkum—Potassium Transport in Enteromorpha 71

T A B L E 3. Thermodynamics of K+ accumulation for Enteromorpha grown in seawater andACBSW medium

In Part A the number of observations (n) on which the membrane potentials are based is shown inbrackets. The membrane potential values are taken from Ritchie and Larkum (1984a). An error of±20 mV has been allowed to take into account a possible vacuolar potential. In Part B the directionof presumptive active transport is shown in brackets. Assymetrical error-bars of the calculatedelectrochemical potentials have been ignored and replaced by the larger of the + and — 95%confidence limits. The tonoplast potential was unknown and so only an upper estimate of theelectrochemical potential across the tonoplast has been made.

Part A: The Nernst equilibrium potential

Activity(Ko

+ mMolal)

Plants in seawater

Plants in ACBSW

Activity(Kc

+ mMolal)

9-59 232 ±30

0-49 187 ±30

forK +

Activity £K*fto £K.Y(P

(KT+ mMolal) (mV) (mV)'

330 + «

82±11

- 8 0 + 4 - 8 9 + 5

-150 + 5 -126 + 4— 4 —3

Plasmalemma membrane potential observed (ific,o)Seawater ACBSW medium

Light - 5 4 ± 5 m V ( n = 20) -Dark - 3 0 ±5 mV (n = 16)

Part B. Electrochemical potential of K +

Conditions

Plants in seawaterLightDark

Plants in ACBSWLightDark

Plasmalemma(kJmor1)

2-5 ±0-58 (in)4-8 ±0-58 (in)

4-3 ±0-91 (in)5-9 ±0-91 (in)

-105±8 mV(n= 16)- 8 9 ± 8 mV(n= 16)

i Tonoplast(k Jmor 1 )(upper estimate)

2-80 ±0-58 (in)2-80 ±0-58 (in)

4-2 ±0-61 (in)4-2 ±0-61 (in)

(mVT

- 9 ± 6

-24 + 6

vacuolar flux in the case of plants in low-salinity (ACBSW) medium (equation 2). The uptakerates were not significantly different in the light and dark and so could be combined to yieldan overall mean uptake rate into the vacuole (Table 2).

Energetics of K+ transport

Table 3 (part A) shows the Nernst equilibrium potential (EK*Xo) for K + in the cytoplasmand vacuole respectively, compared to the bathing medium for plants in seawater and in lowsalinity medium (data from Table 1). In seawater, the Kc

+/K^ and K^/K^ systems areboth close to, but significantly different from,electrochemical equilibrium (£K\M> = lAi.o); K +

is actively transported across the plasmalemma into the cytoplasm. In low-salinity medium,K + closely approaches electrochemical equilibrium across the plasmalemma, particularly inthe light, but is sufficiently different for active transport of K + to be invoked. The K +

equilibrium potential between the vacuole and the bathing electrolyte {EK*Vfi) is notsignificantly different from the observed membrane potential in the light but is differentin the dark.

72 Ritchie and Larkum—Potassium Transport in Enteromorpha

T A B L E 4. Permeability correction factor and apparent K+ permeability in Enteromorpha inseawater and ACBSW

The permeability correction factor function calculated by the procedure of Ritchie and Larkum (1984a,equation 5) is used to take into account the electrical potential gradient across the plasmalemmawhen calculating the apparent permeability shown in Part B. The tonoplast potential has beenassumed to be zero.

Part A. Permeability correction factor

Conditions

Seawater, Light

Dark

ACBSW, Light

Dark

Part B. Apparent

Conditions

Seawater, Light

Dark

ACBSW, Dark

Dark

Membrane potentialftCi0) (mV)

-54±5

-30±5

-1O5±8

- 8 9 ± 8

permeability of K +

2-02 ±046

l-39±O30

100 ±030

100 ±030

3.5Q + O50J - 0 4 3j.92 + 0-24

- 0 2 1151 -tl9 . 3 4 + 2-6y - 2 0

(nm s"1)

2 . 6 7 + O65

9 2 9 _ 3-31

Table 3 (part B) presents the calculated electrochemical potential, in kJ mol ', requiredto maintain K + in the steady state across the plasmalemma (equation 2), in the light anddark. The direction of active transport is shown in brackets. In seawater, K + closelyapproaches electrochemical equilibrium across the plasmalemma, particularly in the lightbut is sufficiently different for active transport of K+ to be invoked. In ACBSW, about 50 kJmol~ * is required to maintain the cytoplasmic K+ in equilibrium with the bathing electrolytein the light and dark.

Electrochemical equilibrium across the tonoplast is more in doubt because no data isavailable for the tonoplast potential but it is likely to be between —20 mV and +20 mV(Hope and Walker, 1975). An estimate of the largest probable expenditure of energy at thetonoplast can be made. In plants in seawater £Kv,c is close to zero mV (£v c = E,o — £co).Taking a tonoplast potential of + 20 mV as the least thermodynamically favourable case,the electrochemical potential of K + across the tonoplast can be calculated to be about+ 29 ± 6 mV or 2-8 ± 0-58 kJ mol ~' for active transport into the vacuole from the cytoplasm.In low-salinity plants £K >,<: 1S about —24 mV and so could either be close to or morethan —44 mV from electrochemical equilibrium. If the tonoplast potential is —20 mV this

Ritchie and Larkum—Potassium Transport in Enteromorpha 73

leads to an upper limit of energy expenditure at the tonoplast of 4-2 ±0-61 kJ mol"1 onK+ transport.

Apparent permeabilityTable 3 shows that K+ appears to be actively accumulated across the plasmalemma of

Enteromorpha, hence efflux of K+ out of the cells would be passive. Table 4 (part A) shows theevaluation of the permeability correction factor / ( + l,A\ji) (Ritchie and Larkum, 1984a,equation 5) and the membrane potential values from Ritchie and Larkum (1984a, Table 1).The apparent permeability of K+ across the plasmalemma of plants in seawater andACBSW media is shown in Table 4 (part B) and were calculated as previously describedfor Cl~ (Ritchie and Larkum, 1984a). The permeabilities have large errors because themembrane potential, the fluxes and the [Kc

+] each have large relative errors.The apparent permeability of K+ at the plasmalemma of seawater plants is about 2-7 nm

s"1; and is significantly higher in the light than in the dark (t-test, P < O05). PNaV.o w a s

found previously to be about 0002 nm s"1 in the dark (Ritchie and Larkum, 1984b); thusJ V / * V is about 00003.

The apparent permeability of K+ in low-salinity Enteromorpha plants is higher than inseawater plants. PK*^0 is independent of light, unlike the case of seawater plants. Takingthe results from light and dark experiments mean PK^ gives a mean of 12 + 2 nm s~l.N̂a*Ao is 0-117±0-O47 nm s"1 (Ritchie and Larkum, 1984b) leading to a J W / / V ratio

of about 0005 and hence the discrimination for K+ over Na+ is much less at lowersalinity.

DISCUSSION

In a variety of studies of marine algae it has been concluded that although K+ in the cell isoften close to thermodynamic equilibrium, nevertheless K+ is actively accumulated acrossthe plasmalemma by a light-stimulated pump (Gutknecht and Dainty, 1968; Raven, 1976).Often these claims for light stimulation appear to be based on inadequate statisticalevidence.

Enteromorpha intestinalis in seawater typically exhibited a single intracellular 4 2K+ ex-change phase (Figs 1 and 2) and gave mean intracellular (K/] of 349 mMolal, in agreementwith the Ca(NO3)2 washing method and previous findings, in Enteromorpha (Black andWeeks, 1972). Similar observations have been made for Ulva (West and Pitman, 1967b;Dickson, Wyn-Jones, and Davenport, 1980) and Porphyra (Reed and Collins, 1980, 1981a;Reed, Collins, and Russell, 1981). Thus, in seawater the transfer of 4 2K+ across theplasmalemma was the rate-limiting step. The tonoplast flux must be several times theplasmalemma flux (West and Pitman, 1967b).

Enteromorpha discriminated between 8 6Rb+ and *2K+ (Fig. 2) in a similar way to Ulva(West and Pitman, 1967a) and Porphyra (Reed and Collins, 1981 a). Using the same reasoningas the latter studies, we have calculated from the intracellular compartmentation of 8 6Rb+ acytoplasmic concentration [Kc

+] of 279 mMolal and a vacuolar concentration [Kv+] of

405 mMol. This estimate of the [Kc+] in the cytoplasm is higher than that calculated in

Porphyra (183 mol m"3) and is also high in comparison with that measured in marine giantalgal cells, where the ion content of the cytoplasm can be analysed separately from that of thevacuole (Raven, 1976, Table 6.4; Hope and Walker, 1975, Table 5.5; Hastings and Gutknecht,1976). The K+ concentration is probably an overestimate because such a [K^] would haveinhibitory effects on enzymes unless they were specifically adapted to such K+ concentra-tions (Wyn-Jones, Storey, and Pollard, 1977; Wyn-Jones, Brady, and Speirs, 1979).

74 Ritchie and Larkum—Potassium Transport in Enteromorpha

The plasmalemma fluxes were measured using efflux and uptake methods (Tables 1 and 2,respectively). Both methods showed that in seawater , the K+ flux was greater in the lightthan in the dark. The higher efflux rates, in comparison with influx rates probably due todifferent culture batches. Seawater efflux experiments using CCCP (Ritchie and Larkum,1985) were conducted at the same time as the uptake experiments and had control 4 2K +

efflux rates comparable to the uptake data. The wide variation in 4 2 K + efflux rates seemsto have been neglected in many previous studies.

The efflux rate of 560 nmol m ~2 s ~' in Enteromorpha in seawater is about 2-3 times thatfound in Porphyra (Reed and Collins, 1980,1981a; Reed et al., 1981). Using the data of Westand Pitman (19676) we estimate that Ulva lactuca, although a close relative of Enteromorpha,has a plasmalemma flux of only about 111 nmol m~2 s" ' . The plasmalemma surface areasused by Black and Weeks (1972) in their study of Enteromorpha are probably erroneous(Ritchie, 1982; Ritchie and Larkum, 1984a) and so their estimates of 4 2 K + fluxes willnot be considered. The plasmalemma flux in Enteromorpha is high compared to fluxesfound in many other marine algae (Gutknecht and Dainty, 1968; Raven, 1976; Hope andWalker, 1975).

There was little effect of light on 4 2 K + fluxes in Enteromorpha in seawater and nosignificant effect in the case of low-salinity plants. At best, the effect of light is a 50%stimulation of the K + flux. Many authors have claimed a light effect on K + fluxes or [K + J inalgae (e.g. Viva; West and Pitman, 19676; Dickson et al, 1980; Scott and Hayward, 1953a, b,c, 1954, 1955; Scott, DeVoe, Hayward, and Craven, 1957; Hydrodictyon; Raven, 1967a, b,1968, 1969; Rybova, Janacek, and Slavikova, 1972). A case for a direct light-stimulation of aK + pump can only be made for a few algae (e.g. Halicystis; Graves and Gutknecht, 1976); inmost algae it is probable that light acts indirectly through its effects on the power availableby controlling the size of the ATP pool (Reid and Walker, 1983). Light also had little effect onthe membrane potential, / V , intracellular K + ([Kj+]) or the intracellular compartmenta-tion of K + .

Although Enteromorpha has a wide salinity tolerance, these are the first published 4 2K +

exchange data on Enteromorpha at low salinities. The mean intracellular [K (+] of

Enteromorpha in ACBSW medium was 118 mMolal based on 4 2 K + labelling; this is notsignificantly different to that measured using the Ca(NO3)2 rinsing method (Table 1). Incontrast to the 4 2 K + experiments on seawater-grown plants, the low-salinity plantsexhibited two exchange phases from which [Kc

+] was estimated to be 222 mMolal and[Kv

+] as 82 mMolal. The plasmalemma efflux (221 nmol m~2 s"1) was lower than that ofplants in seawater and was insensitive to light. No significant difference in total K + content([Kj+]), cytoplasmic ([Kc

+]) or vacuolar K + ([Kv+]) was found between light and dark

experiments. Reed et al. (1981) found that Porphyra exhibited a single 4 2 K + exchange phasein salinities as low at 25% seawater, so that [Kc

+] and [Kv+] could not be estimated, but the

mean intracellular [K,+] was found to be 94 mMolal. The [K^] is comparable to that foundin Nitellopsis which lives in brackish water (MacRobbie and Dainty, 1958) and that found infreshwater algae such as Hydrodictyon and Chora (Raven, 1976; Hope and Walker, 1975).

K + was in the steady state in the vacuole of Enteromorpha grown at low salinity. It is likelythat the cytoplasm was also in the steady state. Although the steady-state condition is lesscertain in plants in seawater a similar analysis has been attempted there. The plants used inthis work were from healthy laboratory cultures and so we were not using senescent materialas may be the case with field-collected material. The Nernst criterion shows that K + is closeto electrochemical equilibrium across the plasmalemma and tonoplast in both seawater andlow-salinity medium (Table 3). The membrane potential values (^, J shown in Table 3 arepresumed to be the plasmalemma potential (^co) where ^ v o = ipco. However, the actual

Ritchie and Larkum—Potassium Transport in Enteromorpha 75

\]/e o could differ by ± 20 mV based on the known tonoplast potentials of most giant algalcells (Hope and Walker, 1975).

Contrary to the opinion of Black and Weeks (1972), we could not ascertain by opticalmicroscopy whether a micro-electrode was in the cytoplasm or vacuole. In plants inseawater, in the light, the observed distribution of K+ in the cytoplasm and the bathingmedium would be in electrochemical equilibrium if the actual plasmalemma membranepotential (4>c,o)was about —75 mV; hence in the light there is a marginal case for active K +

transport. The vacuolar K + ([Kv+]) has an equilibrium potential of about —88 ± 5 mV

(Table 3) suggesting active uptake across the tonoplast and/or the plasmalemma. The powerconsumption of active K + uptake at the tonoplast would be so high that this possibility isunlikely (Ritchie, 1982) and it is more likely that active K + uptake occurs at theplasmalemma in both the light and dark.

In low-salinity plants (Table 3) the evidence for active K+ transport is stronger than inseawater plants. Active uptake of K + across the plasmalemma needs to be invoked both inthe light and dark since the difference between the observed membrane potential and theequilibrium potential (EK*^0) is more than —30 mV. In low-salinity, active transport ofchloride also occurs at the plasmalemma (Ritchie and Larkum, 1984a) and the equilibriumpotentials of K+ and Cl~ across the tonoplast (£K*ViC and Ea-Vf) are close to each other. It istherefore, likely that the tonoplast potential (i/\>c) is near to -I-10 mV.

It is difficult to come to any conclusions about active transport of K + at the tonoplastwithout measurements of the tonoplast potential. If active transport of K + does occur at thetonoplast, then an upper estimate of the electrochemical potential across the tonoplastwould be of the order of 3-4 kJ mol ~' in plants in seawater and low salinity (Table 3, part B).

The thermodynamics of K + , Na + (Ritchie and Larkum, 1984b) and Cl" (Ritchie andLarkum, 1984a) are closely comparable to those found in Porphyra purpurea (Reed andCollins, 1980, 1981a, b; Reed et a/., 1981). Both algae are noted for their wide salinitytolerance. Both algae have thick cell walls and share a very low intracellular Na + and fluxand high intracellular K + and flux. Thus a K^/Na0

+u, exchange pump does not seem

to be important in these algae.The free energy of hydrolysis of ATP in the intracellular environment can be taken as near

to 50 kJ m o P l (Nicholls, 1982; Reid and Walker, 1983) thus active transport of K+ inEnteromorpha could easily be driven directly or indirectly by ATP. An ATP driven K +

uptake pump is an example of primary active transport, an example of an indirect mechanismwould be a H + / K + symport driven by an ATP driven proton extrusion pump (secondaryactive transport) (Nicholls, 1982). From a thermodynamic point of view primary andsecondary active transport mechanisms are equivalent.

If 2K+ are transported per ATP hydrolysed, the thermodynamic efficiency of such pumpswould be low. For example, K + uptake into the cytoplasm, in plants in low-salinity medium,in the dark requires the greatest input of free energy, about 5-9±0-91 kJ mol"1 (Table 3),implying a thermodynamic efficiency of only about 30%. Current evidence suggests mostATPases transport two ions per ATP hydrolysed (Slayman, Lu, and Shane, 1970; Slayman,Long, and Lu, 1973; Walker and Smith, 1975; Smith and Walker, 1981; Gradmann andKlemke, 1974; Harold, 1977; Poole, 1978; Coster, 1981) but the well-known K + /Na +

exchange ATPase of animal cells normally transports 2K+ /3Na+ and, furthermore, thestoichiometry of this pump has been shown to be variable (Sarkadi and Tosteson, 1979).

The apparent permeability of cells to K+ in Enteromorpha in both seawater and inACBSW are comparable to those found in other algae (Hope and Walker, 1975; Raven,1976) for example Nitellopsis (MacRobbie and Dainty, 1958), Nitella (MacRobbie, 1962),Griffithsia (Findlay, Hope, and Williams, 1970) and previous work on Enteromorpha (Black

76 Ritchie and Larkum—Potassium Transport in Enteromorpha

and Weeks, 1972) and on squid nerves (Katz, 1966). The apparent permeability of K+ is notvery different in low-salinity (ACBSW) medium. However, /VAO of sea water plants issignificantly higher in the light than in the dark whereas PK'^o is independent of light inACBSW plants (Table 4).

ACKNOWLEDGEMENTSThis work was partially supported by a research grant from the University of Sydney. One ofthe authors (R.J.R.) would like to thank the Department of Veterans Affairs via the SoldiersChildren Education Scheme for financial assistance. The work described in this paperformed part of a Ph.D. thesis by Raymond J. Ritchie submitted to the University of Sydney1982.

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