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III. Energy-Dependent and Active Ion Transport

III. Energy-Dependent and Active Ion Transport

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are killed. Because the electrical potential is negative inside relative to outside, cations are drawn in and anions are repelled. Thus, cations may exist

in cells at much higher concentrations than outside, but when the concentration (activity) and electrical gradients (both are physical driving forces)

are both taken into account, the ion may possess the same electrochemical

potential on both sides of the membrane. In this situation, an ion can exist

at a higher concentration inside than outside due to the electrical driving

force, and this would be called an energy-dependent transport since energy

is necessary for maintaining the electrical potential. But, transport per se

would be passive since only physical driving forces acted on the ion. In

this situation, energy expenditure would be indirect.

Active transport is a special type of energy-dependent transport. Since

active transport is defined as the movement of an ion against its electrochemical gradient, this type of transport is an “uphill” process, and it must

be directly coupled to an energy releasing reaction.

Carriers, permeases, translocases, transporters, and porters are terms

used to describe substances which reside in membranes and aid the solute

in moving across the membrane, presumably through an association or

binding. In this discussion the term carrier is used since it is most common

in the literature on transport in plants. The two main characteristics of

carrier-mediated transport are saturating kinetics and specificity. Active

transport, as defined above, exhibits these characteristics and is therefore

believed to be carrier mediated.

Passive transport that is energy-dependent also frequently exhibits saturation kinetics and specificity and is therefore also thought to involve carriers. Such transport is frequently termed facilitated diffusion.Still another

type of carrier-mediated, but passive, transport phenomenon is exchangediffusion. In this type of transport, a carrier can only traverse the membrane when complexed with a specific ion. For example, when a radioactive ion is moved across the membrane from outside to inside by this type

of carrier and then released, the carrier will not return until it binds a

similar ion. There is a small chance the carrier will recombine with the

labeled ion; thus the return trip is likely to be with a similar, but nonradioactive ion. Thus, exchange-diffusion results in a bidirectional transport,

and no net transport occurs.

In summary, the term energy-dependent transport accurately describes

all transport that depends on metabolism, and it frequently leads to accumulation ratios significantly greater than 1, but the transport itself may be

either active (i.e., against the electrochemical gradient) or passive (down

the electrochemical gradient). Both types of transport may be carriermediated. Facilitated diffusion and exchange-diff usion are merely descriptive terms for carrier-mediated passive transport.







From the previous discussion, it is apparent that information about the

electrical potential difference across cell membranes is necessary in order

to determine whether a specific ion is actively transported. Numerous reports describe experimental techniques for measuring membrane potentials

of plant cells, and the problems encountered in these studies have been

clearly discussed by Dainty (1962). In the most common technique a

Ag/AgCl microelectrode with a salt bridge ( 3 N KC1) is inserted into

the cytoplasm or vacuole and connected through an electrometer to a similar electrode placed in the ambient solution. The major problems in these

studies are that electrodes frequently break due to the impervious nature

of the plant cell wall, and it is difficult to know the cellular location of

the electrode tip. In addition, protoplasm plugs the salt bridge, membranes

reseal over the tip of the electrode, junction potentials occur at the electrode tip, etc. The problems notwithstanding, reliable data are now available about membrane potentials for several species of algae (MacRobbie,

1970) and for a few higher plants (Higinbotham, 1973). The electrical

potential difference across the plasma membrane is in the range of -60

mV to -200 mV (cytoplasmic phase negative) and the electrical potential

difference across the tonoplast is relatively small, being only 0 to -20

mV with the cytoplasm being negative relative to the vacuole.

Two types of analysis have been used with plant tissue for determining

whether ions are actively transported. One of these employs the Nernst

equation which relates ion activity (concentration) difference across cell

membranes to the electrical potential difference across the cell membrane

under equilibrium conditions. The other analysis used is the flux-ratio comparison which was developed independently by Ussing (1949) and Teorell

(1949). In this analysis the ratio of the actual measured flux rates

(influx/efflux) of a particular ion is compared to the ratio of the predicted

passive flux rates (influx/efflux) . Both the Nernst and flux-ratio analysis

predict the passive distribution of ions. If the actual ion concentrations

or flux rates differ significantly from that predicted on the basis of the

physical driving forces, this is interpreted as proof that the ion under consideration is actively transported.

1. Nernst Equation Analysis

The Nernst equation is based on equilibrium conditions (see Dainty,

1962; Higinbotham, 1973), and for any ion ( j ) that is passively distributed across a semi-permeable membrane the electrochemical potential

( p ) of the ion is the same on the two sides of the membrane i.e., p j 0 = pi',



where o and i refer to an outside solution and an inside solution, respectively (as an example, the plasma membrane separating the outside solution,

0, from the cytoplasmic solution, i ) . The electrochemical potential of an

ion consists of two major components-the chemical potential ( R T In a )

and the electrical potential ( z F E ) where R is the gas constant, T is the

absolute temperature, a is the activity of the ion,* z is the valence of the

ion, F is the Faraday, and E is the electrical potential. Thus, ii, =


RT In a, and since the electrochemical potential of a passively

distributed ion is the same on both sides of the membrane at equilibrium, it

follows that z,FEjo RT In aj0 = z,FE,'

RT In a j l . Then E,' - E , O =

AE = R T / z , F In al0/a,', which is the Nernst equation. For our considerations, we will only be concerned with monovalent ions and if we assume a

temperature of 2OoC and convert to the common logarithm and also substitute concentration ( c ) for activity ( a ) , the Nernst equation simplifies to

AE = 2 5 8 log c,o/cli.

The Nernst equation, in this application, simply indicates the equilibrium relationship between an electrical potential difference across a membrane and the concentration or accumulation ratio, i.e., cjl/c,O, for an

ion which is passively distributed across the membrane. For example, an

electrical potential difference ( A E ) , at 20°C, of -58 mV would result

in an equilibrium concentration ratio of 10 for cations and 0.1 for anions.

A AE of -1 16 mV, which is close to that found for higher plants (Etherton and Higinbotham, 1960; Higinbotham et al., 1964), would yield an

equilibrium concentration ratio of 100 for cations and 0.01 for anions.

If the latter situation were, in fact, found to be true in an experiment,

i.e., a AE of -116 mV, external cation and anion concentrations of say

1 mM and internal cation and anion concentrations of 100 mM and 0.01

mM, respectively, one would have to conclude that both the cation and

anion were passively distributed. The cation transport would be described

as an energy-dependent transport because the accumulation ratio was

greater than 1 and this was made possible by energy expenditure in maintaining the electrical potential difference across the membrane. Although

anion transport is also energy-dependent, this would not be recognized as

such if only the internal and external anion concentrations were known.

When major deviations occur between the actual internal concentrations

and those that are predicted based on the AE, and quasi-equilibrium conditions exist, this is considered proof that the ion is actively transported.

In the above example where the AE was - 116 mV, if the cation was maintained at a concentration exceeding 100 mM, this would indicate that the




* In practice, concentrations are frequently substituted for the activities, but direct

estimates of activities, using ion selective microelectrodes, have been used (Coster,

1966; Vorobiev, 1967; Etherton, 1968).



ion was being actively pumped into the cell. If, on the other hand, the

cation was maintained at a concentration less than 100 mM, this would

indicate the ion was actively transported out of the cell. Similarly, internal

anion concentrations in excess of 0.01 mM would indicate an inward directed active transport, whereas concentrations less than 0.01 mM would

indicate an active extrusion.

The Nernst equation has been used commonly for evaluating ion fluxes

in algae (MacRobbie, 1970) but only rarely for higher plants. The most

thorough studies using this approach with higher plants is that of Etherton

and Higinbotham (1960) and Higinbotham et al. (1964, 1967). They

have reported that the interior of cells of oats, peas, and corn is electrically

negative by 80 to 115 mV relative to an external solution of 0.1 mM KCl

(Etherton and Higinbotham, 1960). Most of this electrical potential difference is across the plasma membrane; only a small electrical potential difference across the tonoplast seems to be a general feature of plant cells (MacRobbie, 1970). Subsequent studies using roots and coleoptiles of oats, and

roots and stems of peas, indicated that all anions (i.e., C1-, NO,-, H,PO,-,

and SO,") were actively transported into the cells of these tissues (Higinbotham et al., 1964, 1967). Although the cations (Na+, Ca2+,Mg ,+) entered the cells by an energy-dependent process (i.e., accumulation ratios

were in excess of 1) , they appeared to be actively secreted. In other words,

the internal concentrations of these cations were less than they should have

been based on the passive driving forces. However, because of the low

permeability coefficients of the divalent cations they are probably excluded

rather than actively secreted (Higinbotham et al., 1967). Potassium appeared to be close to electrochemical equilibrium, and therefore passively

distributed, when the external salt concentration was low, but actively extruded from the cells, like the other cations, when the external salt concentration was high.

Based on other studies using the Nernst equation, there seems to be

no conflict with the conclusion that anions are actively absorbed by higher

plant cells (Dunlop and Bowling, 1971; Gerson and Poole, 1972); however, there is some uncertainty with regard to the nature of cation transport. For example, in red beet tissue (Poole, 1966) and in corn roots

(Dunlop and Bowling, 1971) K' i6 actively transported inward. Both K+

and Na' have been reported to be actively absorbed by sunflower roots

(Bowling and Ansari, 1971, 1972). In the pitcher of the pitcher plant

(Nepenthes henryana), Na+ was actively transported into the cells

(Nemctk et al., 1966), but K+ appeared to be passively distributed. Thus,

using the Nernst equation, it is not clear whether cations are actively

pumped or passively distributed across the membranes of higher plant




One of the major difficulties in using the Nernst equation for evaluating

ion transport is that this equation is based on an equilibrium condition,

and this does not exist in actively metabolizing cells. For this reason, the

flux-ratio equation, which does not require equilibrium conditions, has also

been used for evaluating the driving forces acting on ions.

2 . Flux-Ratio Analysis

The flux-ratio, or Ussing-Teorell, equation for passive ion transport was

developed independently by Ussing ( 1949) and Teorell (1949). The equation is as follows:




Jout cJLe x p ( z , F E / R T )

Where I,,,and Jollt represent radioactive tracer influx and efflux, respectively, across a particular membrane, c,” and c,’ are the concentrations

of ion j on the two sides of the membrane, and the other terms were

identified in the previous section. By measuring E , c j 0 , and c I 1one can

which is the ratio of the rates of ion influx

calculate a value for J,I1/J~,,lt

and efflux which would occur passively, i.e., in response to the existing

concentration and electrical driving forces. This passive flux-ratio can then

be compared to the actual flux-ratio of the ion in question, and if the ion

is in fact moving passively the calculated and measured flux-ratios will be

identical. If they are different, this indicates that an additional driving

force is acting on the ion, and this is taken as evidence that the ion is

actively transported, and depending on whether the measured J l n / J o r l t

ratio is greater or smaller than the calculated J,,I/Jo,ltratio, the direction

of active transport can be ascertained.

It is difficult, however, to obtain the necessary information to make the

flux-ratio test. One needs to know the rates of influx and efflux of a specific

ion across both the plasma membrane and tonoplast as well as the concentrations of the ion in the external solution, cytoplasm, and vacuole. As

difficult as it seems though, great strides have been made in the last 10

years in determining these values. MacRobbie and Dainty (1958a,b) and

Diamond and Solomon (1959) were the first to use what is called a “compartmental analysis” technique for estimating these various parameters in

plant cells. Since the estimation of the various fluxes across the two membranes and the concentrations of the ion in the cytoplasm and vacuole

is crucial to the flux-ratio test, the basis of the compartmental analysis

will be considered first.

a . Compartmental Analysis. When certain assumptions are made (MacRobbie, 1971), one can estimate specific ion fluxes at the plasma mem-



brane and tonoplast by evaluating the kinetics of movement of a radioactive ion into and out of a tissue. The assumptions in this analysis have

been discussed by Cereijido and Rotunno (1970), and the most important

ones are (1 ) that the cells consist of compartments whose fluxes are in

series rather than in parallel, i.e., vacuole + cytoplasm e cell wall F? outside solution as opposed to say vacuoleeoutside solution and cytoplasm e outside solution, ( 2 ) that the vacuole compartment is much

larger than the cytoplasmic compartment, and ( 3 ) that the tissue is in a

steady-state with respect to ion content, i.e., the chemical ion content of

the tissue does not change during the course of “loading” and “unloading”

the tissue with the radioisotope. MacRobbie ( 1971) has provided a most

valuable assessment of these assumptions and the method in general, and

the reader should consult this review for a critical evaluation. Also, the

papers by Cram (1968a) and Pallaghy and Scott (1969) provide a most

lucid derivation of the equations involved in calculating the various flux

rates and ion contents of various compartments.

Experimentally, plant tissue in a steady-state condition3 is exposed to

a nutrient solution containing radioisotope for a given time (generally several hours) and then transferred to a solution containing an identical nutrient solution, except for the radioisotope, for an additional period of time

(again for several hours). Entry of isotope into the tissue is monitored

during the “loading” period, and its exit from the tissue is monitored during the “unloading” period. The nutrient solution must be replaced frequently during the “unloading” period in order to minimize reabsorption

of the radioisotope.

The loss of radioisotope from the tissue appears to be best described

as a series of first-order reactions (Cereijido and Rotunno, 1970), which

can be resolved by plotting the efflux data logarithmically as a function

of time-as in Fig. 1A [first-order reactions yield a straight line when the

loss of reactant (ion in this case) is plotted logarithmically as a function

of time, i.e., log A = (--k/2.303)t

A , with the slope equal to

--k/2.303 and the intercept, A,,, being the initial amount of reactant]. The

radioisotope loss from tissue is initially rapid but develops with time into

a slow, linear rate of loss. This loss is interpreted as the loss from a specific

cell compartment, and by extrapolating this line to t = 0, one has an estimate of the amount of radioisotope in this cell compartment at the beginning of the “unloading” period. Depending on the “loading” time, the


‘ A net accumulation of ion occurs in low salt roots, thus a steady-state does

not exist and such roots are not amenable to the compartmental analysis. The

negligible efflux under these conditions (Epstein, 1973) must be attributed t o the

low ion content of the roots.



amount of radioisotope in this compartment can represent as much as 90%

of the total radioisotope in the tissue, and it is argued that the only cell

compartment likely to contain this much of the label is the vacuole (Pitman, 1963; Cram, 1968a; MacRobbie, 1971).

From the slope of the linear component one can calculate the first-order

rate constant (slope = - k / 2 . 3 0 3 ) from which one can then determine the






from vccuoIes

Loss trom cytoplasm

Time (mid


FIG. 1. Logarithm of the efflux of radioactive ions from roots as a function

of time. ( A ) The linear portion of the curve is interpreted as the loss of ions

from cell vacuoles. Extrapolation of this slope to time zero gives the amount

of radioactivity in the vacuoles at the beginning of efflux. I , is the radioactivity

in vacuoles at time zero divided by the specific radioactivity of the “loading”

solution, So. (B) Same as in plot A, but with the radioactive content of the vacuoles

subtracted. Linear component is interpreted as the loss of radioactivity from the

cytoplasm. I , is the radioactivity at time zero divided by the specific radioactivity

of the “loading” solution, So.

half-time (t,,2 = 0.693/k) for radiois.otope loss from the vacuoles. For

higher plants the reported half-times for ion efflux from vacuoles ranges

from about 50 to 2000 hours (Pitman, 1963; Pierce and Higinbotham,

1970), depending on the tissue employed, the ion, and the experimental

conditions. The most common values, however, range from about 50 to

100 hours. So, for most plants, under steady-state conditions, replacement

of half the ions in the vacuoles would require 2-4 days. For complete turn-



over or replacement of ions in vacuoles, it would take in excess of 10-20

days (about 98 % loss occurs in 5 half-times) , a period considerably longer

than most ion absorption experiments.

The amount of radioisotope remaining in the vacuoles at any time can

be directly determined from the extrapolated line (Fig. l A ) , and if these

values are subtracted from the total radioactivity in the tissue at the various

times, one obtains the loss of radioactivity from the other parts of the cell.

When these values are plotted logarithmically as a function of time (Fig.

l B ) , an initial rapid loss of radioisotope from the tissue develops into another linear component. This first-order component is believed to represent

the loss from the second largest cell compartment, the cytoplasm (MacRobbie and Dainty, 1958a; Pitman, 1963; Etherton, 1967; Cram, 1968a;

Pierce and Higinbotham, 1970). The rate constant for ion loss from the

cytoplasm is determined from the slope and then the half-time for radioisotope loss from the cytoplasm can be calculated. Half-times for ion losses

from cytoplasm of higher plant cells vary from about 8 to 250 minutes,

depending on the tissue, the ion, and the experimental conditions (Pierce

and Higinbotham, 1970). The most common cytoplasmic half-times are

between 10 and 40 minutes. Thus, for most situations, 98% of the ions

in the cytoplasm would turnover in 50 to 200 minutes. Accordingly, absorption periods of these durations would result in the specific radioactivity

of the cytoplasm being similar to that of the external solution.

By continuing to subtract the slow component of radioisotope loss from

that in the tissue it is possible to distinguish the loss of radioisotope from

2 additional compartments. Macklon and Higinbotham ( 1970) have suggested these compartments correspond to cell walls and a surface film. The

half-times for isotope loss from cell walls and the surface film is only a

few minutes.

The apparent ion content of the cytoplasm can be estimated directly

from the radioisotope data if the loading time exceeds 5 times the cytoplasmic half-time (i.e., cpm at intercept of second plot divided by the external specific radioactivity-referred to as I,. below). The ion content of

the cytoplasm in micromoles/gram is readily converted to concentration

by assuming 1 g of tissue equal 1 ml (or an appropriate correction factor

can be applied for the water content of the tissue) and dividing by the

volume of the cytoplasm. The ion content of vacuoles (QV) can be estimated by determining the total ion content of the tissue by chemical

methods and subtracting the ion content of the cell walls and cytoplasm

that is estimated from the radioactivity data (again the loading time should

be in excess of 5 times the cytoplasmic half-time for this to be a reliable

estimate). The ion content of vacuoles can be converted to concentration

by knowing the volume of the cell occupied by the vacuole.



The various flux rates (Cram, 1968a; Pallaghy and Scott; 1969; Pierce

and Higinbotham, 1970; MacRobbie, 1971) can then be determined from

the following equations:


= kclc


= kclc








4-J v - o



where J is the flux rate, o is the outer phase, c is cytoplasm, v is the

vacuole, k, and k , are the efflux rate constants for cytoplasm and vacuole,

respectively, and they are obtained from the slope of the “unloading”

curves, I , and I , are the apparent radioisotope contents based on the

t = o intercepts from the cytoplasmic and vacuole efflux curves divided by

the external specific activity, t , , is the absorption or “loading” time and

Q, is the vacuole content of ion as described above.

The compartmental analysis is obviously of great interest since it provides estimates of ion fluxes across separate membranes and ion contents

of different cell compartments which heretofore had not been made. However, it is still not clear whether this analysis is valid for higher plant cells.

Both Pitman (1963) and Cram (1968a) have obtained evidence in support of the series model for higher plant cells; however, MacRobbie ( 1969,

1971) has found that the series model does not accurately describe ion

fluxes in Nitella cells. In Nitella, the transfer of C1- to vacuoles occurs

more rapidly than predicted by the series model. Pallaghy et al. (1970)

and Neirinckx and Bange (1971 ) have also reported that ion influx in

corn and barley roots, respectively, is not consistent with the series model.

It has also been found that the “unloading” curves for excised roots have

an anomalous shape at low external concentrations which does not occur

for roots of intact plants (Pallaghy et al., 1970; Weigl, 1971). This anomaly may represent the transfer of ions into the xylem and out into the external solution through the cut ends of the roots; Pitman (1971) estimates

that this type of ion transfer may account for as much as 75% of the

radioisotope in the wash-out solutions. Although various discrepancies and

problems have arisen in the compartmental analysis, it represents the only

procedure at the present time for even qualitatively estimating ion fluxes

at both the plasma membrane and tonoplast and the concentrations of ions

in the cytoplasm and vacuole of higher plant cells.

6. Experimental Flux-Ratio Comparisons. Several attempts have been

made to test for passive or active ion transport in higher plant cells using



the flux-ratio equation in association with the flux rates, and ion concentrations that are estimated using the compartmental analysis procedure (Pitman, 1963; Pitman and Saddler, 1967; Etherton, 1967; Cram, 1968a;

Scott et al., 1968; Poole 1969; Pallaghy and Scott, 1969; Macklon and

Higinbotham, 1970; Pierce and Higinbotham, 1970). Although the actual

conclusions based on this test must be made with reservations due to the


A. Ion Fluxes across the Plasma Membrane of Oat Coleoptilesn,*











1 .a








1 R5













B. Ion Fluxes across the Tonoplast of Oat Coleoptilesamb





J v --re












?1 . s















Adapted from Pierce and Higinbotham (1970).

X 10lO/g/secand Q values are millimolar. The calculated

= c,O/c,' exp ( z , F E / R T ) ]

flux ratios were based on the Ussing-Teorell equation [Jln/Jout

using measured E values of - 110 mV across the plasma membrane and 0 mV across the

tonoplast, and activity coefficients of 0.81 for all ions in the outside solution and 0.77-for

all ions in the cytoplasm and vacuole. Qc and Q, were calculated assuming that the cytoplasm and vacuole represented 3.5 and R9% of the cell volunie.


* Units for flux rates are moles

uncertainty of the compartmental analysis, it does provide a tentative appraisal of the driving forces involved in transport. For higher plant cells,

the most complete analysis to date using this approach was made by Pierce

and Higinbotham (1970) for K+, Na+, and C1- transport in oat coleoptiles.

Some of their data are summarized in Table I.

At the plasma membrane, the calculated passive flux-ratio for C1- was

0.0016 and the measured flux-ratio was 4.6. From this it would certainly

appear that C1- is actively pumped inward across the plasma membrane

of the oat coleoptile cells. Cram (1968a) obtained similar results with



carrot tissue. The passive flux-ratio for Na+ at the plasma membrane of

the coleoptile cells (Table IA) was estimated to be 68 and the measured

value only 2.6 indicating an active efflux of this ion. Active Na+ efflux at the

plasma membrane has also been reported for pea roots (Etherton, 1967),

barley roots (Pitman and Saddler, 1967), and broadbean roots (Scott et al.,

1968). Conclusions about Kt transport at the plasma membrane are

variable. In the oat coleoptile, K appears to be actively pumped inward

(Table IA, Pierce and Higinbotham, 1970). A similar conclusion was

reached for barley roots (Pitman and Saddler, 1967) and for pea epicotyl

cells (Macklon and Higinbotham, 1970). However, Scott et al. (1968)

concluded that only nonvacuolated cells of barley roots transported K+ inward at the plasma membrane; Kt was passively distributed in the mature

vacuolated cells. Etherton (1967) also found K+ to be passively distributed

across the plasma membrane of pea roots. In contrast to these results,

Poole (1969) found a low flux-ratio for K+ at the plasma membrane of

beet cells, and he suggested that 'an active K+ extrusion or possibly a K+

exchange-diffusion phenomenon was involved.

At the tonoplast of the oat coleoptile cells, active transport of both K+

and Na+ into vacuoles appeared to occur (Table IB). Chloride was passively distributed. In carrots, however, chloride appears to be actively

pumped across the tonoplast into vacuoles (Cram, 1968a, 1969a).

Although the number of investigations concerning active and passive

ion transport in cells of higher plants are limited, some generalizations,

based on both the Nernst equation and flux-ratio analysis, appear to be

valid. Chloride, and anions in general, are actively transpgrted inward

across the plasma membrane. Cations, for the most part, enter cells by

an energy-dependent process, but the movement is down the electrochemical gradient. Under certain situations the cations also appear to be actively

transported across the plasma membrane but the direction of transport

is variable; sometimes it is into the cell and other times it is out of the

cell. In most cases, Na+ seems to be actively secreted. Observations on

tonoplast ion fluxes are very limited, but it seems that active transport

may also occur at this membrane. Additional investigations of the individual ion fluxes across both the plasma membrane and tonoplast and tests

for passive or active transport are urgently needed and a complete picture

of ion transport will not be possible until such studies have been


3 . Origin of the Electrical Potential Difference

The electrical potential difference across biological membranes has generally been considered to be only a diffusion potential (Dainty, 1962);

however, recent studies indicate that this may not be so (Higinbotham,

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III. Energy-Dependent and Active Ion Transport

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