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IV. Kinetics and Selectivity of Ion Absorption

IV. Kinetics and Selectivity of Ion Absorption

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ION ABSORPTION BY PLANT ROOTS



181



range (0-1 mM) does saturate and can be adequately described by Michaelis-Menten kinetics (for barley roots the K,,, for K+ is about 0.01 mM

and V,,,.,,is about 10 pmoles per gram fresh weight per hour), and they

have referred to this as mechanism I (see Fig. 2 ) . But in the high concentration range (1-50 mM), a true saturation does not occur and the absorption isotherm is not smooth, but “bumpy” or heterogeneous (Elzam et al.,

1964; Epstein and Rains, 1965; Epstein, 1966; see Fig. 2). Epstein

refers to absorption in this range as mechanism 11.



Ion Concentration (mM)



FIG. 2. Diagrammatic representation of the velocity of ion influx into roots as

a function of the external ion concentration.



The most thorough characterization of the kinetics of ion absorption

has been for K+, Na+, and C1- in barley roots (Epstein, 1972a, 1973).

Mechanism I is highly specific for K+ and C1-, providing Ca2+is present

in the absorption solution (Jacobson et al., 1950; Epstein, 1961). Other

monovalent cations and anions, except for Rb+ and Br-, respectively, have

little effect on K+ and C1- absorption. This specificity is lost in the absence

of Ca2+and ions and like Na+ or H+ will interfere with K+ absorption.

K+ absorption by mechanism I is virtually unaffected by the counter anion;

K+ absorption rates are the same in the presence of the rapidly absorbed

C1- ions or the slowly absorbed SO,’- ion (Epstein et al., 1963). Mechanism I1 is quite different. It is more specific for Na’ than K+, and Ca2+

inhibits the absorption of these ions (Rains and Epstein, 1967a,b). The

nature of the anion was also shown to be important in the high concentra-



182



T. K. HODGES



tion range; K+ or Na’ absorption from K,SO, or Na,S04 is much less than

from KC1 or NaCI. The kinetics and selectivity of K+ and Na+ transport

in roots of several other species is similar to that found for barley roots;

however, some differences do exist (Fried and Broeshart, 1967; Gauch,

1972; Epstein, 1972a, 1973).

The complexity of ion absorption kinetics and the various ion interactions in roots are not easily interpreted, and they have led to considerable

debate (Laties, 1969; Epstein, 1973). Bacteria have been suggested to

be responsible for absorption at low concentrations (Barber, 1966), but

this seems to have been ruled out using roots grown and handled aseptically (Epstein, 1968, 1972b). Absorption in the high concentration range

has been suggested to be a concentration-dependent diffusion phenomena

(see Laties, 1969). However, at least for C1-, transport over the entire

concentration range (up to 60 mM) is against the electrochemical gradient

(Gerson and Poole, 1972) and therefore active. Also, metabolic inhibitors

impair transport at both low and high external ion concentrations (Toni

and Laties, 1966a; Rains and Epstein, 1967a; Ordin and Jacobson, 1955).

Torii and Laties (1966a) and Luttge and Laties (1966, 1967) concluded

that mechanism I is located at the plasma membrane and mechanism I1

is located at the tonoplast. Welch and Epstein (1968, 1969), on the other

hand, concluded that mechanism I and I1 were located at the plasma membrane. Recently Nissen ( 1971) has suggested that a single mechanism,

rather than two, is responsible for the transport of sulfate into barley roots

and leaves, and that this mechanism resides in the plasma membrane. He

suggested that the carrier undergoes phase transformations, and changes

in K,,, and V,,la., at certain critical external concentrations. From these

various interpretations, it is apparent that the kinetics of ion transport are

complex and that controversy exists.

However, if one considers the kinetic studies along with the evidence concerning which ions are actively transported (see Section 111), three observations seem to be particularly important. First, all anions are actively

pumped inward across the plasma membrane and some cations, like Na+,

seem to be actively extruded across the plasma membrane (Higinbotham

et al., 1967). Second, all the cations and all the anions exhibit influx kinetics that are of the pseudo-saturation type. This results in the kinetic constants, K , and V,,,,, continually increasing as the external ion concentration increases, and in certain cases both cations and anions exhibit the

“bumps” in the absorption isotherm (Hagen and Hopkins, 1955; Hagen

et al., 1957; Fried and Noggle, 1958; Fried et al., 1961; Noggle and Fried,

1960; Hodges and Vaadia, 1964; Bange and Meijer, 1966; Weigl, 1967,

1970; Penth and Weigl, 1969; Nissen, 1971; Leonard and Hodges, 1973)

[also see reviews by Laties (1969) and Epstein (1973) for their compre-



ION ABSORPTION BY PLANT ROOTS



183



hensive studies]. Thus, active inward anion transport appears to involve

carriers (two or more?) and cation entry also appears to involve carriers

(two or more?), but the cation transport is supposedly passive. Furthermore, since some studies show cations to be actively extruded, this presumably means that cation efflux is carrier mediated. This, of course, implicates a rather bewildering array of different carriers. The third important

observation is that the selectivity of transport changes as the external ion

concentration increases (Epstein, 1966, 1973). All these experimental observations, complex as they seem, can be readily accounted for by a single

ion carrier. The basis for this view is the similarity between ion transport

kinetics and cooperativity kinetics of single enzymes (Monod et al., 1963;

Koshland, 1970).

Kinetics of single enzymes are basically of 3 types (Koshland, 1970).

One is the standard Michaelis-Menten kinetics which exhibit a true saturation when velocity is plotted as a function of substrate concentration (i.e.,

v vs S) . A second is positive cooperativity kinetics which yields a sigmoidal

or S-shaped curve in the v vs S plot. The third is negative cooperativity

kinetics which shows a pseudo-saturation curve in the v vs S plot. A fourth

type of kinetics yields a “bumpy” isotherm in the v vs S plot, but this

is believed to be primarily due to negative cooperativity with a minor

component of positive cooperativity kinetics (Levitski and Koshland,

1969).

A model to account for these various types of kinetics has been proposed by Koshland (1970). The model assumes a multisubunit enzyme

with each subunit possessing identical binding sites for a particular ligand

(e.g., substrate, activator, or inhibitor). Ligand binding to the first subunit,

however, induces a conformational change (Gerhart and Pardee, 1962;

Koshland, 1969) which alters or distorts the other subunits sufficiently to

change the kinetic characteristics of their binding sites; with negative cooperativity the K,, increases, and with positive cooperativity the K , decreases. Thus, with negative cooperativity, binding of the first ligand makes

it more difficult for the second, binding of the second makes it more difficult

for the third, etc. So, with increasing ligand concentration the velocity of

the reaction approaches a maximum rate or saturation more slowly than

a reaction where no subunit interaction occurs, i.e., one possessing Michaelis-Menten kinetics. Because of the decreasing affinity of the subunits

as the ligand concentration increases, Lineweaver-Burk plots of negative

cooperativity kinetics are always concave downward curves rather than

straight lines, and similarly Eadie, Hofstee plots yield curves instead of

the straight lines typical of a Michaelis-Menten reaction (Koshland, 1970).

The kinetics of ion transport are virtually identical to the kinetics described as negative cooperativity for single enzymes (Koshland, 1970).



T. K. HODGES



184



Lineweaver-Burk and Eadie, Hofstee plots of absorption data always yield

curves when a large concentration range is examined (see all the references

cited previously that show the pseudo-saturation kinetics and also see Fig.

3 ) . Thus, as the external ion concentration increases, the K , and V,,,

for ion transport continually increases. Additionally, it has been found that

the Hill coefficient (Hill, 1910) for 4*K+transport into oat roots is 0.56

(Leonard and Hodges, 1973), which is characteristic of negative cooperativity kinetics (Koshland, 1970). Thus, it would seem that the seemingly



I



I



I



I



FIG. 3. Eadie, Hofstee plot of “K’ influx into oat roots. (From Leonard and

Hodges, 1973.)



complex kinetics of ion transport could be accounted for by a single carrier

consisting of several identical subunits. In keeping with the Koshland

(1970) model, ion binding to the first subunit would induce a conformational change, and this would distort the other subunits such that their

affinity for ions would decrease. This would bring about the increasing

Km’s as the ion concentration is increased. Overall, the negative cooperativity model accounts for ion transport kinetics, but due to the “bumps”

in the absorption isotherms, an additional component of positive cooperativity could also be involved. This model for explaining ion transport kinetics is quite similar to the phase transformation model of Nissen (1971),

however, the present model provides an explanation for the changing kinetic patterns which occur.



ION ABSORPTION BY PLANT ROOTS



185



A single carrier having binding sites with varying affinities for ions could

also account for the selectivity of ion transport. For example, it has been

shown that the specificity of alkali ion binding to anionic groups of glass

electrodes is determined by the electric field strength of the negative site

(Eisenman, 1961, 1962), and this has been shown to be valid for all kinds

of anionic groups on many types of substances including resins, clays, artificial membranes, and numerous biological systems (Diamond and Wright,

1969). Eisenman showed that for the 5 alkali cations, only eleven selectivity

sequences (out of a possible 120 permutations) occur as the binding site

field strength changes from a low to a high value. These sequences are

as follows:

I

I1

I11

IV

V

VI

VII

VIII

IX

X

XI



> Na+ > Li+

> Na+ > Li+

Rhf > K+ > Cs+ > Na+ > 12

K+ > Rb+ > Cs+ > Na+ > Li+

K+ > Rh+ > Na+ > CY+> Li+

K+ > Na+ > Rb+ > Cs+ > Li+

Na+ > K+ > Rhf > Cs+ > Li+

Na+ > K+ > Rb+ > Li+ > Cs+

Na+ > K+ > Li+ > Rb+ > Cs+

Na+ > Li+ > K+ > RIP > Cs+

Li+ > Na+ > K+ > Rb+ > Cs+

Cs+ > Rb+ > K+

Rbf > Cs+ > K+



Sequence I is in the order of increasing apparent hydrated size and Sequence XI is in the order of increasing nonhydrated size. In each of the

intermediate sequences, one pair of cations shifts positions. The basis for

these selectivity patterns is the relative free energy differences between

ion: site and ion :water electrostatic interactions. Thus, the cation preferred

by a specific negative site will be that cation which experiences the greatest

decrease in free energy when its nearest neighbor becomes the negative

site rather than water. When the negative site has a very strong electric

field strength, the free energy differences between ion :site and ion: water

interactions is such that the cation with the smallest ionic radius will be

preferred and thus the selectivity pattern will be that shown in Sequence

XI. But, if the negative site has a weak electric field strength the free energy differences between ion:site and ion:watcr interactions is such that the

largest nonhydrated cation, which has the lowest free energy of hydration,

will be preferred and the order of selectivity will be that shown in Sequence

I. Thus, when the electric field strength of the negative site is very weak,

Sequence I is preferred, and as the electric field strength of the negative

site increases, the order of specificity of ion binding progressively shifts

until at very high electric field strengths, Sequence XI is the order of preference. Eisenman also showed that the selectivity for H+ relative to the



186



T. K. HODGES



alkali ions is also dependent on the field strength of the binding site. At

high field strengths, H+ is preferred over the alkali ions and at low field

strengths, the alkali ions are preferred over H+.

The basis for selective ion transport by plants is almost sure to reside

in the electric field strength of the ion binding sites and in shifts in the

electrical strength of the sites. For example, in barley roots, the preference

for K+ over Na+ at low external concentrations and the reverse at high

external concentrations (Epstein, 1961; Rains and Epstein, 1967a,b) indicates that the field strength of the binding site increases as the external

ion concentration increases. A change in the field strength of the binding

sites could be brought about by conformational changes in carrier subunits

as described previously. The shift from a low to a high field strength, with

sequential ion binding, would also bring about a continually increasing

preference for Ht by the binding sites. This could account for the continually decreasing affinity, or increase in apparent K,, for the alkali cations

as the external concentration increases.

There are numerous reports for the selectivity of ion absorption by

plants where two or three of the alkali cations have been considered (Collander, 1941; Fried and Broeshart, 1967), but very few where all five of

the alkali cations have been studied. Steward and Mott (1970) reported

an order of alkali cation preference by carrot cells that corresponds to

Sequence VI. In a very thorough study, Jacobson et al. (1960) determined

the effect of both pH and Ca2+on the absorption of the alkali ions by

barley roots. In the presence of Ca2+and at pH 7 the preferred sequence

was Kt > Rb+ > Na’ > Cst > Lit (i.e., Sequence V ) . As the pH decreased

to 3, in the presence of Ca2+,a shift occurred such that the preferred sequence was Rb+ > K > Cs’ > Nat > Li+ (i.e., Sequence 111). It was not

possible to determine from their figures whether Sequence IV occurred. This

effect of H+ on changing the selectivity pattern of transport is analogous

to the effect of Ht on the selectivity pattern of alkali ion binding to glass

electrodes (see Diamond and Wright, 1969). The effect of increasing the

proton concentration is to reduce the “effective” negative charge strength

of the binding site. The effect of Ca2+on the selectivity is complex because

of its association with various negative charges, but it too appeared to alter

the “effective” field strength of the transport sites since selectivity was

shifted from the higher to the lower sequences in barley roots (Jacobson

et al., 1960). The CaZt-induced specificity for Kt over Nat at low external

concentrations (Epstein, 1973) would also be consistent with this interpretation. An alteration in the “effective” field strength by Ca2+might also

be the basis for the anomalous effect of Ca2+on Kt absorption in barley

and wheat roots (Hiatt, 1970a,b) as well as the promotive effect of CaZt

on K+ transport under some conditions (Viets, 1944; Overstreet et al.,



ION ABSORPTION BY PLANT ROOTS



187



1952; Kahn and Hanson, 1957; Epstein, 196 1 ), and its inhibition in others

(Handley et al., 1965; Elzam and Hodges, 1967).

Eisenman ( 1965) has further shown that halide specificity by fixed positive charges is also governed by the relative free energy differences between

ion:site and ion: water interactions. Of the 24 possible selectivity sequences, only seven occur commonly. Diamond and Wright (1969) have

cited some deviations from the main seven orders and discuss why these

sometimes occur. The normal seven are as follows:

I



I1



I11

IV

V

VI



VII



I- > Br- > CIBr- > I- > C1Br- > CI- > ICI- > Rr- > IC1- > Br- > FCI- > F- > Br-



F- > CI- > Rr-



> F> F> F>P

> I> I> J-



A site with a very strong electric field would prefer the ion having the

smallest nonhydrated radius, F-, and Sequence VII would be the order of

specificity. A very low field strength site would prefer the most nonhydrated

ion, I-, and Sequence I would be preferred.

In roots, C1- is generally absorbed at about the same rate as Br- (Epstein, 1953; Boszormenyi and Cseh, 1961, 1964) or slightly faster (Elzam

and Epstein, 1965) and both are absorbed more rapidly than either F(Venkateswarlu et al., 1965) or I- (Boszormenyi and Cseh, 1964). Probably one of the sequences from I1 to V correctly describes the normal

selectivity pattern for halide absorption by roots. Whether the ion concentration, pH or Ca+affect the selectivity sequence is unknown.

The concept of a single cation carrier and a single anion carrier is supported by observations that the total cation or total anion absorption is

generally constant from salt solutions that vary in the proportions of the

cations or anions (Bear and Prince, 1945; Jacobson et al., 1960; Jackson

and Stief, 1965; Hiatt, 1968, 1969, 1970a,b; Pitman et al., 1968). The

clearest example of this is for K and Na' absorpion by barley roots (Jackson and Stief, 1965). They found the combined rates of K' and Na' transport were constant even though the individual rates of K+ and Na+ transport were different. These experiments were conducted in the absence of

Ca2+.However, Hiatt ( 1970a,b) obtained virtually the same results when

Ca2+was present.

A single carrier for cation influx would not account for the active efflux

of cations, such as sodium (see Section 111), unless the carrier functions

in an exchange manner. There is evidence that cation influx is in exchange

for H i in low salt roots (Jackson and Adams, 1963; Jacobson et al., 1950;

Pitman, 1969) and that a Kt/Kt exchange becomes more prominent as



188



T. K. HODGES



salt saturation is approached (Pitman, 1969; ,also see Poole, 1969). Active sodium efflux might be accomplished by a general cation carrier if

exchange is involved and if the carrier has a high affinity for Na+ when

the site(s) faces the cytoplasm. All that would be needed to accomplish

an active Na+ efflux would be for the carrier site, when facing the cytoplasm, to have a sufficiently high electric field strength that Na+ is preferred

over the other cytoplasmic ions. Jeschke (1970) and Pitman and Saddler

(1967) have presented evidence that a K+ influx-Na+ efflux does occur

in barley roots. This type of exchange, however, is not as specific as it

PlOIlnO



MambrOM

Li < Csc No< Rb c K

Hiqh Ccncentration

Li c Csc Rb.r K c Na



I



I

CATION

CARRIER

H+. No'



,TF



OH-, HCO;



ANION

CARRIER



FI < I c Br < CI



FIG.4. A model depicting a single cation exchange carrier and a single anion

exchange carrier in the plasma membrane of root cells.



is for the active K+/Na+ exchange reaction of mammalian cells (Skou,

1965). This is also evident from the failure of ouabain to inhibit ion fluxes

in plants (Hodges, 1966; Cram, 1968b). Cram (1968b) did observe a

slight inhibition of Na+ efflux by ouabain, but there was no evidence that

the Na+ efflux was tightly coupled to K influx. Thus, energy-dependent

cation exchange does occur, but it is not highly specific. It is suggested,

however, that it could have sufficient specificity to account for the active

Na+efflux at the plasma membrane.

A model depicting a single cation carrier and a single anion carrier is

shown in Fig. 4. Both carriers are considered to carry out an energy-dependent exchange of external for internal ions. The approximate order of

specificity for the alkali cations at low and high external concentrations

is shown. Also, the apparent order of halide specificity by the anion carrier

is shown. As discussed above, there is evidence for energy-dependent ca-



ION ABSORPTION BY PLANT ROOTS



189



tion exchange. However, evidence for energy-dependent anion exchange

is limited. A suggestion of the latter comes from studies that show changes

in organic acid levels in the cell when the absorption rates of cations and

anions are different (Ulrich, 1941; Jacobson and Ordin, 1954; Hiatt and

Hendricks, 1967; Hiatt, 1967a,b). Thus, when inorganic anion absorption

exceeds inorganic cation absorption, an anion/HCO,- exchange is a strong

possibility. Also, as will be discussed in Section VI, a HC0,- influx coupled

to a OH- efflux on the anion carrier is suggested when cation absorption

exceeds anion absorption. Finally, the exchange-diffusion of C1- reported

by Cram (1968a) and Cram and Laties (1971) might represent a manifestation of the anion exchange carrier.

One of the strongest arguments against a single carrier for cations is

the shift in preference for Na+ and K+ as a function of aging in stem tissue

(Rains, 1969; Rains and Floyd, 1970) and in red beet tissue (Poole,

1791a,b). At low external concentrations freshly cut bean stem slices

transport Na+ much more rapidly than K+, but after aging 20 hours in

CaS04, K+ is transported more rapidly than Na'. A similar specificity occurs at high concentrations, but it is not as pronounced. Rains (1 969)

and Rains and Floyd (1970) interpret these changes in transport specificity as evidence for the development of a K' carrier that is independent

of the Na+ carrier. A similar phenomenon occurs in the beet tissue (Poole,

1971a,b). K+ transport is more rapid than Na+ transport in slices aged

for 1 day, whereas Na' transport is more rapid than K+ in slices aged for

6 to 7 days. Poole also interprets these data as indicating two separate

carriers. In both tissues, however, an altered specificity of the same carrier

could conceivably account for the results. Many metabolic changes occur

during the washing (aging) period, and alterations in the membrane lipids

or proteins could alter the molecular environment of the carrier to such

an extent that the charge density or field strength of the binding sites would

undoubtedly be altered. Because such a change would alter ion selectivity,

one carrier could probably account for the results.

The model of ion transport proposed here (Fig. 4 ) represents the combination of two widely different concepts, the negative cooperativity concept of Koshland (1970) and the selectivity concept of Eisenman (1961,

1962). Together they account for most, if not all, of the kinetic and selectivity aspects of ion transport in plants.



V.



Energetics of Ion Transport



Aerobic conditions are essential for nutrient absorption by roots. This

has been shown by the pioneering investigations of Steward (1932),



190



T. K. HODGES



Lundegirdh (1934), and Hoagland and Broyer (1936). These studies

were followed by demonstrations of parallels between aerobic respiration

and nutrient absorption (Lundegdrdh and Burstrom, 1933; Ulrich, 1941;

Vlamis and Davis, 1944; Robertson and Turner, 1945; Robertson and

Wilkins, 1948). Finally, the finding that respiratory poisons. such as cyanide, azide, and carbon monoxide inhibited ion absorption clearly established the requirement of aerobic respiration for nutrient absorption by

plant roots (Ordin and Jacobson, 1955).

The precise manner in which aerobic respiration is coupled to ion transport is still uncertain, but many significant observations and interpretations

have been made. The first detailed interpretation of the link or couple between respiration and ion transport was presented by Lundegdrdh and

Burstrom (1933). Lundegdrdh’s concept has been discussed at length

(Lundegirdh, 1939, 1945, 1955), and only the major features will be consided here. In essence, he postulated that during the oxidation of reduced

compounds, electrons were transferred through a chain of cytochromes and

associated with this electron flow was a reversed flow of anions along the

cytochrome chain. Cations were considered to enter cells passively in order

to maintain electrical neutrality. Lundegirdh’s concept met with disfavor

when it was realized that the cytochrome chain was localized in mitochondria and not in the plasma membrane. The concept was also inconsistent

with the finding that the phosphorylation uncoupler, 2,4-dinitrophenol

(DNP), which eliminates the formation of ATP but does not inhibit electron transfer through the cytochrome chain, is a powerful inhibitor of ion

absorption (Robertson et al., 1951 ) . The latter finding was taken as evidence that ATP was the intermediary link between aerobic respiration and

ion transport. This view was further supported by subsequent findings that

arsenate (Ordin and Jacobson, 1955; Higinbotham, 1959; Weigl, 1963,

1964) and oligomycin (Hodges, 1966; Jacoby, 1966; Bledsoe et al.,

1969), which interfere with ATP formation, were also potent inhibitors

of ion absorption. Thus, it would appear that ATP is the actual energy

source for ion transport; however, some observations indicate that this may

not always be so.

Evidence discounting ATP as the source of energy for ion transport has

come from tissues other than roots with the exception of storage root tissue. With aged root tissue of carrots (Atkinson et al., 1966) and beets

(Polya and Atkinson, 1969), various inhibitors such as a nitrogen atmosphere, uncouplers and ethionine (an ATP-trapping agent) did not affect

ion absorption and the levels of ATP in the tissue in parallel. Thus, the

corelation that one would expect if ATP were the energy source did not

exist, and these authors concluded that electron transfer reactions, rather

than ATP, were involved in ion absorption. This interpretation assumes



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