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6 Solute Transport
( ) ( )
–1 for monovalent anions, +2 for divalent cations, and so
∆ m˜ s = m s + RT ln Cs − m s + RT ln Cs
* i * o
on), F is Faraday’s constant (equivalent to the electric
charge on 1 mol of protons), and E is the overall electric (
= RT ln Cs i
− ln C )
s
o
(6.5)
potential of the solution (with respect to ground). The final i
– Cs
term, VjP, expresses the contribution of the partial molal = RT ln
–
volume of j (Vj) and pressure (P) to the chemical potential Cs o
Solute Transport 89
If this difference in chemical potential is negative, sucrose Equation 6.8 shows that ions, such as K+, diffuse in re-
could diffuse inward spontaneously (provided the mem- sponse to both their concentration gradients ([K+]/[K i +]o)
brane had a finite permeability to sucrose; see the next sec- and any electric-potential difference between the two
tion). In other words, the driving force (∆m~s) for solute dif- compartments (Ei – Eo). One very important implication
fusion is related to the magnitude of the concentration of this equation is that ions can be driven passively
gradient (Csi/Cso). against their concentration gradients if an appropriate
If the solute carries an electric charge (as does the potas- voltage (electric field) is applied between the two com-
sium ion), the electrical component of the chemical poten- partments. Because of the importance of electric fields in
tial must also be considered. Suppose the membrane is per- biological transport, m~ is often called the electrochemical
meable to K+ and Cl– rather than to sucrose. Because the potential, and ∆m~ is the difference in electrochemical
ionic species (K+ and Cl–) diffuse independently, each has potential between two compartments.
its own chemical potential. Thus for inward K+ diffusion,
∆ m˜ Κ = m˜ Κ ι − m˜ Κ ο (6.6) TRANSPORT OF IONS ACROSS A
Substituting the appropriate terms from Equation 6.1 into
MEMBRANE BARRIER
Equation 6.6, we get If the two KCl solutions in the previous example are sep-
∆m~s = (RT ln [K+]i + zFEi) – (RT ln [K+]o + zFEo) (6.7) arated by a biological membrane, diffusion is complicated
by the fact that the ions must move through the membrane
and because the electrostatic charge of K+ is +1, z = +1 and as well as across the open solutions. The extent to which
~ = RT ln [K+]i a membrane permits the movement of a substance is called
∆µK + F(Ei – Eo) (6.8)
[K+]o membrane permeability. As will be discussed later, per-
meability depends on the composition of the membrane, as
The magnitude and sign of this expression will indicate the well as on the chemical nature of the solute. In a loose
driving force for K+ diffusion across the membrane, and its sense, permeability can be expressed in terms of a diffusion
direction. A similar expression can be written for Cl– (but coefficient for the solute in the membrane. However, per-
remember that for Cl–, z = –1). meability is influenced by several additional factors, such
90 Chapter 6
as the ability of a substance to enter the membrane, that are electrochemical potential. And unless the membrane is
difficult to measure. very porous, its permeability for the two ions will differ.
Despite its theoretical complexity, we can readily mea- As a consequence of these different permeabilities, K+
sure permeability by determining the rate at which a solute and Cl– initially will diffuse across the membrane at dif-
passes through a membrane under a specific set of condi- ferent rates. The result will be a slight separation of charge,
tions. Generally the membrane will hinder diffusion and which instantly creates an electric potential across the
thus reduce the speed with which equilibrium is reached. membrane. In biological systems, membranes are usually
The permeability or resistance of the membrane itself, how- more permeable to K+ than to Cl–. Therefore, K+ will dif-
ever, cannot alter the final equilibrium conditions. Equilib- fuse out of the cell (compartment A in Figure 6.2) faster
rium occurs when ∆m~j = 0. than Cl–, causing the cell to develop a negative electric
In the sections that follow we will discuss the factors charge with respect to the medium. A potential that devel-
that influence the passive distribution of ions across a ops as a result of diffusion is called a diffusion potential.
membrane. These parameters can be used to predict the An important principle that must always be kept in
relationship between the electrical gradient and the con- mind when the movement of ions across membranes is
centration gradient of an ion. considered is the principle of electrical neutrality. Bulk
solutions always contain equal numbers of anions and
Diffusion Potentials Develop When Oppositely cations. The existence of a membrane potential implies that
Charged Ions Move across a Membrane at the distribution of charges across the membrane is uneven;
Different Rates however, the actual number of unbalanced ions is negligi-
When salts diffuse across a membrane, an electric mem- ble in chemical terms. For example, a membrane potential
brane potential (voltage) can develop. Consider the two of –100 mV (millivolts), like that found across the plasma
KCl solutions separated by a membrane in Figure 6.2. The membranes of many plant cells, results from the presence
K+ and Cl– ions will permeate the membrane indepen- of only one extra anion out of every 100,000 within the
dently as they diffuse down their respective gradients of cell—a concentration difference of only 0.001%!
As Figure 6.2 shows, all of these extra anions are found
immediately adjacent to the surface of the membrane; there
is no charge imbalance throughout the bulk of the cell. In
Compartment A Compartment B
our example of KCl diffusion across a membrane, electri-
Membrane K+ Cl– cal neutrality is preserved because as K+ moves ahead of
Initial conditions: Cl– in the membrane, the resulting diffusion potential
[KCl]A > [KCl]B
retards the movement of K+ and speeds that of Cl–. Ulti-
mately, both ions diffuse at the same rate, but the diffusion
potential persists and can be measured. As the system
moves toward equilibrium and the concentration gradient
collapses, the diffusion potential also collapses.
Diffusion potential exists
until chemical equilibrium The Nernst Equation Relates the Membrane
is reached. – + Potential to the Distribution of an Ion at
Equilibrium
Because the membrane is permeable to both K+ and Cl–
ions, equilibrium in the preceding example will not be
Equilibrium conditions: reached for either ion until the concentration gradients
[KCl]A = [KCl]B
decrease to zero. However, if the membrane were perme-
able to only K+, diffusion of K+ would carry charges across
At chemical equilibrium,
diffusion potential equals the membrane until the membrane potential balanced the
zero. concentration gradient. Because a change in potential
requires very few ions, this balance would be reached
FIGURE 6.2 Development of a diffusion potential and a
charge separation between two compartments separated by
instantly. Transport would then be at equilibrium, even
a membrane that is preferentially permeable to potassium. though the concentration gradients were unchanged.
If the concentration of potassium chloride is higher in com- When the distribution of any solute across a membrane
partment A ([KCl]A > [KCl]B), potassium and chloride ions reaches equilibrium, the passive flux, J (i.e., the amount of
will diffuse at a higher rate into compartment B, and a dif- solute crossing a unit area of membrane per unit time), is
fusion potential will be established. When membranes are
more permeable to potassium than to chloride, potassium
the same in the two directions—outside to inside and
ions will diffuse faster than chloride ions, and charge sepa- inside to outside:
ration (+ and –) will develop. Jo→i = Ji→o
Solute Transport 91
the case with plants and fungi, which may show experimen- ATP synthesis in mitochondria and chloroplasts also
tally measured membrane potentials (often –200 to –100 mV) depends on a H+-ATPase. In these organelles, this transport
that are much more negative than those calculated from the protein is sometimes called ATP synthase because it forms
Goldman equation, which are usually only –80 to –50 mV. ATP rather than hydrolyzing it (see Chapter 11). The struc-
Thus, in addition to the diffusion potential, the membrane ture and function of membrane proteins involved in active
potential has a second component. The excess voltage is pro- and passive transport in plant cells will be discussed later.
vided by the plasma membrane electrogenic H+-ATPase.
Whenever an ion moves into or out of a cell without
being balanced by countermovement of an ion of opposite
MEMBRANE TRANSPORT PROCESSES
charge, a voltage is created across the membrane. Any Artificial membranes made of pure phospholipids have
active transport mechanism that results in the movement been used extensively to study membrane permeability.
of a net electric charge will tend to move the membrane When the permeability of artificial phospholipid bilayers
potential away from the value predicted by the Goldman for ions and molecules is compared with that of biological
equation. Such a transport mechanism is called an electro- membranes, important similarities and differences become
genic pump and is common in living cells. evident (Figure 6.6).
Both biological and artificial membranes have similar
NH2 permeabilities for nonpolar molecules and many small
polar molecules. On the other hand, biological membranes
C N
N C are much more permeable to ions and some large polar
CH molecules, such as sugars, than artificial bilayers are. The
HC C
O O O
N N reason is that, unlike artificial bilayers, biological mem-
branes contain transport proteins that facilitate the passage
–O P O P O P O CH2 O of selected ions and other polar molecules.
O– O– O– Transport proteins exhibit specificity for the solutes they
H H
H H transport, hence their great diversity in cells. The simple
OH OH prokaryote Haemophilus influenzae, the first organism for
Adenosine-5′-triphosphate (ATP 4– ) which the complete genome was sequenced, has only 1743
genes, yet more than 200 of these genes (greater than 10%
The energy required for active transport is often pro- of the genome) encode various proteins involved in mem-
vided by the hydrolysis of ATP. In plants we can study the
dependence of the membrane potential on ATP by observ-
ing the effect of cyanide (CN–) on the membrane potential
–150
(Figure 6.5). Cyanide rapidly poisons the mitochondria,
and the cell’s ATP consequently becomes depleted. As ATP 0.1 mM CN– added
Cell membrane potential (mV)
Permeability of biological membrane (cm s–1) 102 FIGURE 6.6 Typical values for the permeability, P, of a bio-
logical membrane to various substances, compared with
1
O2 those for an artificial phospholipid bilayer. For nonpolar
molecules such as O2 and CO2, and for some small
H2O uncharged molecules such as glycerol, P values are similar
10–2 in both systems. For ions and selected polar molecules,
CO2
including water, the permeability of biological membranes
10–4 is increased by one or more orders of magnitude, because
of the presence of transport proteins. Note the logarithmic
10–6 K+ scale.
Glycerol
Na+
10–8
Cl– port of neutral amino acids may move glycine, alanine, and
10–10 valine with equal ease but not accept aspartic acid or lysine.
In the next several pages we will consider the structures,
functions, and physiological roles of the various membrane
10–10 10–8 10–6 10–4 10–2 1 102
transporters found in plant cells, especially on the plasma
Permeability of lipid bilayer (cm s–1) membrane and tonoplast. We begin with a discussion of
the role of certain transporters (channels and carriers) in
promoting the diffusion of solutes across membranes. We
brane transport. In Arabidopsis, 849 genes, or 4.8% of all then distinguish between primary and secondary active
genes, code for proteins involved in membrane transport. transport, and we discuss the roles of the electrogenic H+-
Although a particular transport protein is usually highly ATPase and various symporters (proteins that transport
specific for the kinds of substances it will transport, its two substances in the same direction simultaneously) in
specificity is not absolute: It generally also transports a driving proton-coupled secondary active transport.
small family of related substances. For example, in plants a
K+ transporter on the plasma membrane may transport Rb+ Channel Transporters Enhance Ion and Water
and Na+ in addition to K+, but K+ is usually preferred. On Diffusion across Membranes
the other hand, the K+ transporter is completely ineffective Three types of membrane transporters enhance the move-
in transporting anions such as Cl– or uncharged solutes ment of solutes across membranes: channels, carriers, and
such as sucrose. Similarly, a protein involved in the trans- pumps (Figure 6.7). Channels are transmembrane proteins
Transported molecule
Energy Low
Electrochemical
Simple diffusion potential gradient
FIGURE 6.7 Three classes of membrane transport proteins: channels, carriers, and
pumps. Channels and carriers can mediate the passive transport of solutes across
membranes (by simple diffusion or facilitated diffusion), down the solute’s gradient
of electrochemical potential. Channel proteins act as membrane pores, and their
specificity is determined primarily by the biophysical properties of the channel.
Carrier proteins bind the transported molecule on one side of the membrane and
release it on the other side. Primary active transport is carried out by pumps and
uses energy directly, usually from ATP hydrolysis, to pump solutes against their
gradient of electrochemical potential.
Solute Transport 95
(A) (B)
+
+
K+ +
+
+
S1 S2 S3 S4 S5 S6
N C
CYTOPLASM
FIGURE 6.8 Models of K+ channels in plants. (A) Top view of channel, looking through the pore of
the protein. Membrane-spanning helices of four subunits come together in an inverted teepee with
the pore at the center. The pore-forming regions of the four subunits dip into the membrane, with a
K+ selectivity finger region formed at the outer (near) part of the pore (more details on the struc-
ture of this channel can be found in Web Essay 6.1). (B) Side view of the inward rectifying K+ chan-
nel, showing a polypeptide chain of one subunit, with six membrane-spanning helices. The fourth
helix contains positively-charged amino acids and acts as a voltage-sensor. The pore-forming
region is a loop between helices 5 and 6. (A after Leng et al. 2002; B after Buchanan et al. 2000.)
that function as selective pores, through which molecules tuned to the prevailing conditions. Thus the ion perme-
or ions can diffuse across the membrane. The size of a pore ability of a membrane is a variable that depends on the mix
and the density of surface charges on its interior lining of ion channels that are open at a particular time.
determine its transport specificity. Transport through chan- As we saw in the experiment of Table 6.1, the distribu-
nels is always passive, and because the specificity of trans- tion of most ions is not close to equilibrium across the
port depends on pore size and electric charge more than on membrane. Anion channels will always function to allow
selective binding, channel transport is limited mainly to anions to diffuse out of the cell, and other mechanisms are
ions or water (Figure 6.8). needed for anion uptake. Similarly, calcium channels can
Transport through a channel may or may not involve function only in the direction of calcium release into the
transient binding of the solute to the channel protein. In cytosol, and calcium must be expelled by active transport.
any case, as long as the channel pore is open, solutes that The exception is potassium, which can diffuse either
can penetrate the pore diffuse through it extremely rapidly: inward or outward, depending on whether the membrane
about 108 ions per second through each channel protein. potential is more negative or more positive than EK, the
Channels are not open all the time: Channel proteins have potassium equilibrium potential.
structures called gates that open and close the pore in K+ channels that open only at more negative potentials
response to external signals (see Figure 6.8B). Signals that are specialized for inward diffusion of K+ and are known
can open or close gates include voltage changes, hormone as inward-rectifying, or simply inward, K+ channels. Con-
binding, or light. For example, voltage-gated channels open versely, K+ channels that open only at more positive poten-
or close in response to changes in the membrane potential. tials are outward-rectifying, or outward, K+ channels (see
Individual ion channels can be studied in detail by the Web Essay 6.1). Whereas inward K+ channels function in
technique of patch clamp electrophysiology (see Web Topic the accumulation of K+ from the environment, or in the
6.2), which can detect the electric current carried by ions opening of stomata, various outward K+ channels function
diffusing through a single channel. Patch clamp studies in the closing of stomata, in the release of K+ into the xylem
reveal that, for a given ion, such as potassium, a given or in regulation of the membrane potential.
membrane has a variety of different channels. These chan-
nels may open in different voltage ranges, or in response to Carriers Bind and Transport Specific Substances
different signals, which may include K+ or Ca2+ concen- Unlike channels, carrier proteins do not have pores that
trations, pH, protein kinases and phosphatases, and so on. extend completely across the membrane. In transport
This specificity enables the transport of each ion to be fine- mediated by a carrier, the substance being transported is
96 Chapter 6
initially bound to a specific site on the carrier protein. This contrast, the H+/K+-ATPase of the animal gastric mucosa
requirement for binding allows carriers to be highly selec- pumps one H+ out of the cell for every one K+ in, so there
tive for a particular substrate to be transported. Carriers is no net movement of charge across the membrane. There-
therefore specialize in the transport of specific organic fore, the H+/K+-ATPase is an electroneutral pump.
metabolites. Binding causes a conformational change in the In the plasma membranes of plants, fungi, and bacteria,
protein, which exposes the substance to the solution on the as well as in plant tonoplasts and other plant and animal
other side of the membrane. Transport is complete when endomembranes, H+ is the principal ion that is electro-
the substance dissociates from the carrier’s binding site. genically pumped across the membrane. The plasma mem-
Because a conformational change in the protein is brane H+-ATPase generates the gradient of electrochemi-
required to transport individual molecules or ions, the rate cal potentials of H+ across the plasma membranes, while
of transport by a carrier is many orders of magnitude the vacuolar H+-ATPase and the H+-pyrophosphatase
slower than through a channel. Typically, carriers may (H+-PPase) electrogenically pump protons into the lumen
transport 100 to 1000 ions or molecules per second, which of the vacuole and the Golgi cisternae.
is about 106 times slower than transport through a channel. In plant plasma membranes, the most prominent pumps
The binding and release of a molecule at a specific site on are for H+ and Ca2+, and the direction of pumping is out-
a protein that occur in carrier-mediated transport are sim- ward. Therefore another mechanism is needed to drive the
ilar to the binding and release of molecules from an active uptake of most mineral nutrients. The other impor-
enzyme in an enzyme-catalyzed reaction. As will be dis- tant way that solutes can be actively transported across a
cussed later in the chapter, enzyme kinetics has been used membrane against their gradient of electrochemical poten-
to characterize transport carrier proteins (for a detailed dis- tial is by coupling of the uphill transport of one solute to
cussion on kinetics, see Chapter 2 on the web site). the downhill transport of another. This type of carrier-
Carrier-mediated transport (unlike transport through mediated cotransport is termed secondary active transport,
channels) can be either passive or active, and it can transport and it is driven indirectly by pumps.
a much wider range of possible substrates. Passive transport
on a carrier is sometimes called facilitated diffusion, Secondary Active Transport Uses the Energy
although it resembles diffusion only in that it transports sub- Stored in Electrochemical-Potential Gradients
stances down their gradient of electrochemical potential, Protons are extruded from the cytosol by electrogenic H+-
without an additional input of energy. (This term might ATPases operating in the plasma membrane and at the vac-
seem more appropriately applied to transport through chan- uole membrane. Consequently, a membrane potential and
nels, but historically it has not been used in this way.) a pH gradient are created at the expense of ATP hydroly-
sis. This gradient of electrochemical potential for H+, ∆m~ H+,
Primary Active Transport Is Directly Coupled to or (when expressed in other units) the proton motive force
Metabolic or Light Energy (PMF), or ∆p, represents stored free energy in the form of
To carry out active transport, a carrier must couple the the H+ gradient (see Web Topic 6.3).
uphill transport of the solute with another, energy-releas- The proton motive force generated by electrogenic H+
ing, event so that the overall free-energy change is negative. transport is used in secondary active transport to drive the
Primary active transport is coupled directly to a source of transport of many other substances against their gradient
energy other than ∆m~j, such as ATP hydrolysis, an oxida- of electrochemical potentials. Figure 6.9 shows how sec-
tion–reduction reaction (the electron transport chain of ondary transport may involve the binding of a substrate (S)
mitochondria and chloroplasts), or the absorption of light and an ion (usually H+) to a carrier protein, and a confor-
by the carrier protein (in halobacteria, bacteriorhodopsin). mational change in that protein.
The membrane proteins that carry out primary active There are two types of secondary transport: symport
transport are called pumps (see Figure 6.7). Most pumps and antiport. The example shown in Figure 6.9 is called
transport ions, such as H+ or Ca2+. However, as we will symport (and the protein involved is called a symporter)
see later in the chapter, pumps belonging to the “ATP- because the two substances are moving in the same direc-
binding cassette” family of transporters can carry large tion through the membrane (see also Figure 6.10A).
organic molecules. Antiport (facilitated by a protein called an antiporter) refers
Ion pumps can be further characterized as either elec- to coupled transport in which the downhill movement of
trogenic or electroneutral. In general, electrogenic trans- protons drives the active (uphill) transport of a solute in the
port refers to ion transport involving the net movement of opposite direction (Figure 6.10B).
charge across the membrane. In contrast, electroneutral In both types of secondary transport, the ion or solute
transport, as the name implies, involves no net movement being transported simultaneously with the protons is mov-
of charge. For example, the Na+/K+-ATPase of animal cells ing against its gradient of electrochemical potential, so its
pumps three Na+ ions out for every two K+ ions in, result- transport is active. However, the energy driving this trans-
ing in a net outward movement of one positive charge. The port is provided by the proton motive force rather than
Na+/K+-ATPase is therefore an electrogenic ion pump. In directly by ATP hydrolysis.
Solute Transport 97
OUTSIDE OF CELL H+ H+ H+ H+ H+ H+ H+ H+
H+ H+ H+ H+
H+ H+ H+ H+
S + S + S + S +
H+ H+ H H+ H+ H H+ H+ H H+ H+ H
S H+ S H+ S H+ S H+
S H+ S H+ S H+ S H+
H+ H+ H+ H+ H+
S
H+
H+
Plasma
Concentration membrane
gradients S H+
for S and H+ S S
S S S
S S S
H+ S H+ H+ S H+
S S H+ S H+ S S H+ S H+
S S S S
S S S S S S S S
S S S S
H+ H+ H+ H+
S S S S S S S S
S S S S
CYTOPLASM
FIGURE 6.9 Hypothetical model for secondary active transport. The energy that
drives the process has been stored in a ∆m~ H+ (symbolized by the red arrow on the
right in A) and is being used to take up a substrate (S) against its concentration gra-
dient (left-hand red arrow). (A) In the initial conformation, the binding sites on the
protein are exposed to the outside environment and can bind a proton. (B) This
binding results in a conformational change that permits a molecule of S to be
bound. (C) The binding of S causes another conformational change that exposes the
binding sites and their substrates to the inside of the cell. (D) Release of a proton
and a molecule of S to the cell’s interior restores the original conformation of the
carrier and allows a new pumping cycle to begin.
OUTSIDE OF CELL
H+ A H+ B
FIGURE 6.10 Two examples of secondary
High High active transport coupled to a primary pro-
Low
ton gradient. (A) In a symport, the energy
dissipated by a proton moving back into
the cell is coupled to the uptake of one
Electrochemical molecule of a substrate (e.g., a sugar) into
potential the cell. (B) In an antiport, the energy dis-
gradient sipated by a proton moving back into the
cell is coupled to the active transport of a
substrate (for example, a sodium ion) out
of the cell. In both cases, the substrate
High Low Low under consideration is moving against its
gradient of electrochemical potential. Both
H+ A H+ B neutral and charged substrates can be
Electrochemical Electrochemical
potential gradient potential gradient transported by such secondary active
of substrate A CYTOPLASM of substrate B transport processes.
98 Chapter 6
Symporters
Sucrose H+ Amino
H+,Na+ acid
K+ H+
H+
PO43– Antiporter
H+
H+ CYTOSOL
Na+
NO3– pH 7.2
∆E = –120 mV
Plasma
membrane Efflux
Antiporters carrier
Sucrose
ATP
H+ Mg2+
Hexose
Cd2+
ADP + Pi
Sucrose
Ca2+
H+ H+
ATP
H+ H+
3 H+
H+ PC-Cd2+
pumps H+ ATP
Na+ H+ pH 5.5
ADP + Pi ∆E = –90 mV
ABC
ADP + Pi
ATP
VACUOLE Anthocyanin ABC
pH 5.5 H+ GS transporters
pumps 2H+ Tonoplast ABC
ATP ATP
ADP + Pi
H+ Anions,
PPi cations ADP + Pi
ADP + Pi ATP Ca2+
H+ Ca2+ pump
2 Pi
Channels
OUTSIDE OF CELL
K+ Ca2+
Outward Outward
rectifying rectifying
Channels
Typically, transport across a biological membrane is the binding and dissociation of molecules at active sites on
energized by one primary active transport system coupled transport proteins. One advantage of the kinetic approach
to ATP hydrolysis. The transport of that ion—for example, is that it gives new insights into the regulation of transport.
H+—generates an ion gradient and an electrochemical In kinetic experiments the effects of external ion (or
potential. Many other ions or organic substrates can then other solute) concentrations on transport rates are mea-
be transported by a variety of secondary active-transport sured. The kinetic characteristics of the transport rates can
proteins, which energize the transport of their respective then be used to distinguish between different transporters.
substrates by simultaneously carrying one or two H+ ions The maximum rate (Vmax) of carrier-mediated transport,
down their energy gradient. Thus H+ ions circulate across and often channel transport as well, cannot be exceeded,
the membrane, outward through the primary active trans- regardless of the concentration of substrate (Figure 6.12).
port proteins, and back into the cell through the secondary Vmax is approached when the substrate-binding site on the
transport proteins. In plants and fungi, sugars and amino carrier is always occupied. The concentration of carrier, not
acids are taken up by symport with protons. the concentration of solute, becomes rate limiting. Thus
Most of the ionic gradients across membranes of higher Vmax is a measure of the number of molecules of the spe-
plants are generated and maintained by electrochemical- cific carrier protein that are functioning in the membrane.
potential gradients of H+ (Tazawa et al. 1987). In turn, these The constant Km (which is numerically equal to the
H+ gradients are generated by the electrogenic proton solute concentration that yields half the maximal rate of
pumps. Evidence suggests that in plants, Na+ is trans- transport) tends to reflect the properties of the particular
ported out of the cell by a Na+–H+ antiporter and that Cl–, binding site (for a detailed discussion on Km and Vmax see
NO3–, H2PO4–, sucrose, amino acids, and other substances Chapter 2 on the web site). Low Km values indicate high
enter the cell via specific proton symporters. affinity of the transport site for the transported substance.
What about K+? At very low external concentrations, K+ Such values usually imply the operation of a carrier sys-
can be taken up by active symport proteins, but at higher tem. Higher values of Km indicate a lower affinity of the
concentrations it can enter the cell by diffusion through spe- transport site for the solute. The affinity is often so low that
cific K+ channels. However, even influx through channels is in practice Vmax is never reached. In such cases, kinetics
driven by the H+-ATPase, in the sense that K+ diffusion is alone cannot distinguish between carriers and channels.
driven by the membrane potential, which is maintained at Usually transport displays both high-affinity and low-
a value more negative than the K+ equilibrium potential by affinity components when a wide range of solute concen-
the action of the electrogenic H+ pump. Conversely, K+ trations are studied. Figure 6.13 shows sucrose uptake by
efflux requires the membrane potential to be maintained at soybean cotyledon protoplasts as a function of the external
a value more positive than EK, which can be achieved if
efflux of Cl– through Cl– channels is allowed. Several rep-
resentative transport processes located on the plasma mem-
brane and the tonoplast are illustrated in Figure 6.11.
Vmax
1/2 Vmax
other substances (mostly molecules and ions). Active trans-
port utilizes carrier-type proteins that are energized directly
by ATP hydrolysis or indirectly as symporters and
antiporters. The latter systems use the energy of ion gradi-
ents (often a H+ gradient) to drive the uphill transport of
another ion or molecule. In the pages that follow we will
examine in more detail the molecular properties, cellular (Km)
locations, and genetic manipulations of some of these External concentration of
transport proteins. transported molecule
Kinetic Analyses Can Elucidate Transport FIGURE 6.12 Carrier transport often shows saturation
Mechanisms kinetics (Vmax) (see Chapter 2 on the web site), because of
saturation of a binding site. Ideally, diffusion through chan-
Thus far, we have described cellular transport in terms of nels is directly proportional to the concentration of the
its energetics. However, cellular transport can also be stud- transported solute, or for an ion, to the difference in electro-
ied by use of enzyme kinetics because transport involves chemical potential across the membrane.
100 Chapter 6
100
induced in the presence of nitrate in the environment, and
Rate of sucrose uptake
cloned, and several genes for high-affinity K+ carriers have The Plasma Membrane H+-ATPase Has Several
been identified. Further research is needed to determine to Functional Domains
what extent they each contribute to K+ uptake, and how The outward, active transport of H+ across the plasma
they obtain their energy (see Web Topic 6.4). Genes for membrane creates gradients of pH and electric potential
plant vacuolar H+–Ca2+ antiporters and genes for the pro- that drive the transport of many other substances (ions and
ton symport of several amino acids and sugars have also molecules) through the various secondary active-transport
been identified through various genetic techniques (Hirshi proteins. Figure 6.14 illustrates how a membrane H+-
et al. 1996; Tanner and Caspari 1996; Kuehn et al. 1999). ATPase might work.
Plant and fungal plasma membrane H+-ATPases and
Genes for Specific Water Channels Have Been 2+
Ca -ATPases are members of a class known as P-type
Identified ATPases, which are phosphorylated as part of the catalytic
Aquaporins are a class of proteins that is relatively abun- cycle that hydrolyzes ATP. Because of this phosphorylation
dant in plant membranes (see Chapter 3). Aquaporins step, the plasma membrane ATPases are strongly inhibited
reveal no ion currents when expressed in oocytes, but when by orthovanadate (HVO42–), a phosphate (HPO42–) analog
the osmolarity of the external medium is reduced, expres- that competes with phosphate from ATP for the aspartic
sion of these proteins results in swelling and bursting of the acid phosphorylation site on the enzyme. The high affinity
oocytes. The bursting results from rapid influx of water of the enzyme for vanadate is attributed to the fact that
across the oocyte plasma membrane, which normally has a vanadate can mimic the transitional structure of phosphate
very low water permeability. These results show that aqua- during hydrolysis.
porins form water channels in membranes (see Figure 3.6). Plasma membrane H+-ATPases are encoded by a family
The existence of aquaporins was a surprise at first of about ten genes. Each gene encodes an isoform of the
because it was thought that the lipid bilayer is itself suffi- enzyme (Sussman 1994). The isoforms are tissue specific,
ciently permeable to water. Nevertheless, aquaporins are and they are preferentially expressed in the root, the seed,
common in plant and animal membranes, and their expres- the phloem, and so on. The functional specificity of each
sion and activity appear to be regulated, possibly by pro- isoform is not yet understood; it may alter the pH optimum
tein phosphorylation, in response to water availability of some isoforms and allow transport to be regulated in dif-
(Tyerman et al. 2002). ferent ways for each tissue.
OUTSIDE OF CELL
M+ M+ M+ M+ M+ M+ M+
M+ M+
M+
M+ M+ M+ M+ M+ M+
M+ M+ M+ M+ M+
M+
M+ M+ M+ M+ M+ M+
M+ M+ M+ M+
M+
M+
P P
P
Pi
M+
ADP
ATP
CYTOPLASM
FIGURE 6.14 Hypothetical steps in the transport of a cation (the hypothetical M+)
against its chemical gradient by an electrogenic pump. The protein, embedded in the
membrane, binds the cation on the inside of the cell (A) and is phosphorylated by ATP
(B). This phosphorylation leads to a conformational change that exposes the cation to
the outside of the cell and makes it possible for the cation to diffuse away (C). Release
of the phosphate ion (P) from the protein into the cytosol (D) restores the initial con-
figuration of the membrane protein and allows a new pumping cycle to begin.
FIGURE 6.15 Two-dimensional rep-
resentation of the plasma membrane OUTSIDE OF CELL
H+-ATPase. The H+-ATPase has 10
transmembrane segments. The regu- Transmembrane
Plasma membrane
latory domain is the autoinhibitory domains
domain. (From Palmgren 2001.)
G D F
a
d
Although the pH of most plant vacuoles is mildly acidic
V0 (about 5.5), the pH of the vacuoles of some species is much
c
lower—a phenomenon termed hyperacidification. Vacuolar
hyperacidification is the cause of the sour taste of certain
fruits (lemons) and vegetables (rhubarb). Some extreme
examples are listed in Table 6.2. Biochemical studies with
H+ lemon fruits have suggested that the low pH of the lemon
fruit vacuoles (specifically, those of the juice sac cells) is
LUMEN OF VACUOLE due to a combination of factors:
• The low permeability of the vacuolar membrane to
protons permits a steeper pH gradient to build up.
eukaryotes. They are large enzyme complexes, about 750 • A specialized vacuolar ATPase is able to pump pro-
kDa, composed of at least ten different subunits (Lüttge tons more efficiently (with less wasted energy) than
and Ratajczak 1997). These subunits are organized into a normal vacuolar ATPases can (Müller et al. 1997).
peripheral catalytic complex, V1, and an integral membrane
channel complex, V0 (Figure 6.16). Because of their simi-
larities to F-ATPases, vacuolar ATPases are assumed to TABLE 6.2
operate like tiny rotary motors (see Chapter 11). The vacuolar pH of some hyperacidifying plant
Vacuolar ATPases are electrogenic proton pumps that trans- species
port protons from the cytoplasm to the vacuole and generate Tissue Species pHa
a proton motive force across the tonoplast. The electrogenic
proton pumping accounts for the fact that the vacuole is typ- Fruits
Lime (Citrus aurantifolia) 1.7
ically 20 to 30 mV more positive than the cytoplasm, although
Lemon (Citrus limonia) 2.5
it is still negative relative to the external medium. To maintain
Cherry (Prunus cerasus) 2.5
bulk electrical neutrality, anions such as Cl– or malate2– are Grapefruit (Citrus paradisi) 3.0
transported from the cytoplasm into the vacuole through
channels in the membrane (Barkla and Pantoja 1996). Without Leaves
the simultaneous movement of anions along with the pumped Rosette oxalis (Oxalis deppei) 1.3
protons, the charge buildup across the tonoplast would make Wax begonia 1.5
the pumping of additional protons energetically impossible. (Begonia semperflorens)
The conservation of bulk electrical neutrality by anion Begonia ‘Lucerna’ 0.9 – 1.4
transport makes it possible for the vacuolar H+-ATPase to Oxalis sp. 1.9 – 2.6
generate a large concentration (pH) gradient of protons Sorrel (Rumex sp.) 2.6
across the tonoplast. This gradient accounts for the fact that Prickly Pear 1.4 (6:45 A.M.)
the pH of the vacuolar sap is typically about 5.5, while the (Opuntia phaeacantha)b 5.5 (4:00 P.M.)
cytoplasmic pH is 7.0 to 7.5. Whereas the electrical compo- Source: Data from Small 1946.
a The values represent the pH of the juice or expressed sap of each
nent of the proton motive force drives the uptake of anions
tissue, usually a good indicator of vacuolar pH.
into the vacuole, the electrochemical-potential gradient for b The vacuolar pH of the cactus Opuntia phaeacantha varies with the
H+ (∆m m~H+) is harnessed to drive the uptake of cations and time of day. As will be discussed in Chapter 8, many desert succu-
sugars into the vacuole via secondary transport (antiporter) lents have a specialized type of photosynthesis, called crassulacean
acid metabolism (CAM), that causes the pH of the vacuoles to
systems (see Figure 6.11). decrease during the night.
104 Chapter 6
• The accumulation of organic acids such as citric, uole (Bush 1995). Mitochondria and the endoplasmic retic-
malic, and oxalic acids helps maintain the low pH of ulum also store calcium within the cells.
the vacuole by acting as buffers. Calcium efflux from the vacuole into the cytosol may in
some cells be triggered by inositol trisphosphate (IP3). IP3,
Plant Vacuoles Are Energized by a Second Proton which appears to act as a “second messenger” in certain sig-
Pump, the H+-Pyrophosphatase nal transduction pathways, induces the opening of IP3-gated
Another type of proton pump, an H+-pyrophosphatase calcium channels on the tonoplast and endoplasmic reticu-
(H+-PPase) (Rea et al. 1998), appears to work in parallel lum (ER). (For a more detailed description of these sensory
with the vacuolar ATPase to create a proton gradient across transduction pathways see Chapter 14 on the web site.)
the tonoplast (see Figure 6.11). This enzyme consists of a Calcium ATPases are found at the plasma membrane
single polypeptide that has a molecular mass of 80 kDa. (Chung et al. 2000) and in some endomembranes of plant
The H+-PPase harnesses its energy from the hydrolysis of cells (see Figure 6.11). Plant cells regulate cytosolic Ca2+ con-
inorganic pyrophosphate (PPi). centrations by controlling the opening of Ca2+ channels that
The free energy released by PPi hydrolysis is less than that allow calcium to diffuse in, as well as by modulating the
from ATP hydrolysis. However, the vacuolar H+-PPase trans- activity of pumps that drive Ca2+ out of the cytoplasm back
ports only one H+ ion per PPi molecule hydrolyzed, whereas into the extracellular spaces. Whereas the plasma membrane
the vacuolar ATPase appears to transport two H+ ions per calcium pumps move calcium out of the cell, the calcium
ATP hydrolyzed. Thus the energy available per H+ ion trans- pumps on the ER transport calcium into the ER lumen.
ported appears to be the same, and the two enzymes appear
to be able to generate comparable H+ gradients.
In some plants the synthesis of the vacuolar H+-PPase is
ION TRANSPORT IN ROOTS
induced by low O2 levels (hypoxia) or by chilling. This Mineral nutrients absorbed by the root are carried to the
indicates that the vacuolar H+-PPase might function as a shoot by the transpiration stream moving through the
backup system to maintain essential cell metabolism under xylem (see Chapter 4). Both the initial uptake of nutrients
conditions in which ATP supply is depleted because of the and the subsequent movement of mineral ions from the
inhibition of respiration by hypoxia or chilling. It is of inter- root surface across the cortex and into the xylem are highly
est that the plant vacuolar H+-PPase is not found in ani- specific, well-regulated processes.
mals or yeast, although a similar enzyme is present in some Ion transport across the root obeys the same biophysi-
bacteria and protists. cal laws that govern cellular transport. However, as we
Large metabolites such as flavonoids, anthocyanins and have seen in the case of water movement (see Chapter 4),
secondary products of metabolism are sequestered in the the anatomy of roots imposes some special constraints on
vacuole. These large molecules are transported into vac- the pathway of ion movement. In this section we will dis-
uoles by ATP-binding cassette (ABC) transporters. Trans- cuss the pathways and mechanisms involved in the radial
port processes by the ABC transporters consume ATP and movement of ions from the root surface to the tracheary
do not depend on a primary electrochemical gradient (see elements of the xylem.
Web Topic 6.6). Recent studies have shown that ABC trans-
porters can also be found at the plasma membrane and in Solutes Move through Both Apoplast and
mitochondria (Theodoulou 2000). Symplast
Thus far, our discussion of cellular ion transport has not
Calcium Pumps, Antiports, and Channels Regulate included the cell wall. In terms of the transport of small
Intracellular Calcium molecules, the cell wall is an open lattice of polysaccharides
Calcium is another important ion whose concentration is through which mineral nutrients diffuse readily. Because
strongly regulated. Calcium concentrations in the cell wall all plant cells are separated by cell walls, ions can diffuse
and the apoplastic (extracellular) spaces are usually in the across a tissue (or be carried passively by water flow)
millimolar range; free cytosolic Ca2+ concentrations are entirely through the cell wall space without ever entering
maintained at the micromolar (10–6 M) range, against the a living cell. This continuum of cell walls is called the extra-
large electrochemical-potential gradient that drives Ca2+ cellular space, or apoplast (see Figure 4.3).
diffusion into the cell. We can determine the apoplastic volume of a slice of
Small fluctuations in cytosolic Ca2+ concentration dras- plant tissue by comparing the uptake of 3H-labeled water
tically alter the activities of many enzymes, making cal- and 14C-labeled mannitol. Mannitol is a nonpermeating
cium an important second messenger in signal transduc- sugar alcohol that diffuses within the extracellular space
tion. Most of the calcium in the cell is stored in the central but cannot enter the cells. Water, on the other hand, freely
vacuole, where it is taken up via Ca2+–H+ antiporters, penetrates both the cells and the cell walls. Measurements
which use the electrochemical potential of the proton gra- of this type usually show that 5 to 20% of the plant tissue
dient to energize the accumulation of calcium into the vac- volume is occupied by cell walls.
Solute Transport 105
Middle lamella
Tonoplast
Endoplasmic
Cytoplasm
reticulum
Vacuole
Plasmodesma
Protein particles on Desmotubule Protein particles on
inner leaflet of ER with appressed ER inner leaflet of
plasma membrane
FIGURE 6.17 Diagram illustrating how plasmodesmata con-
nect the cytoplasms of neighboring cells. Plasmodesmata
are about 40 nm in diameter and allow diffusion of water
and small molecules from one cell to the next. In addition,
elongation zones. Cells in the root hair zone have com-
the size of the opening can be regulated by rearrange-
ments of the internal proteins to allow the passage of pleted their elongation but have not yet begun secondary
larger molecules. growth. The root hairs are simply extensions of specific epi-
dermal cells that greatly increase the surface area available
for ion absorption.
An ion that enters a root may immediately enter the
Just as the cell walls form a continuous phase, so do the symplast by crossing the plasma membrane of an epider-
cytoplasms of neighboring cells, collectively referred to as mal cell, or it may enter the apoplast and diffuse between
the symplast. Plant cells are interconnected by cytoplasmic the epidermal cells through the cell walls. From the apoplast
bridges called plasmodesmata (see Chapter 1), cylindrical of the cortex, an ion may either cross the plasma membrane
pores 20 to 60 nm in diameter (see Figure 1.27). Each plas- of a cortical cell, thus entering the symplast, or diffuse radi-
modesma is lined with a plasma membrane and contains a ally all the way to the endodermis via the apoplast. In all
narrow tubule, the desmotubule, that is a continuation of cases, ions must enter the symplast before they can enter the
the endoplasmic reticulum. stele, because of the presence of the Casparian strip.
In tissues where significant amounts of intercellular The apoplast forms a continuous phase from the root
transport occur, neighboring cells contain large numbers of surface through the cortex. At the boundary between the
plasmodesmata, up to 15 per square micrometer of cell sur- vascular cylinder (the stele) and the cortex is a layer of spe-
face (Figure 6.17). Specialized secretory cells, such as floral cialized cells, the endodermis. As discussed in Chapters 4
nectaries and leaf salt glands, appear to have high densi- and 5, a suberized cell layer in the endodermis, known as
ties of plasmodesmata; so do the cells near root tips, where the Casparian strip, effectively blocks the entry of water
most nutrient absorption occurs. and mineral ions into the stele via the apoplast.
By injecting dyes or by making electrical-resistance mea- Once an ion has entered the stele through the symplas-
surements on cells containing large numbers of plasmod- tic connections across the endodermis, it continues to dif-
esmata, investigators have shown that ions, water, and fuse from cell to cell into the xylem. Finally, the ion reen-
small solutes can move from cell to cell through these ters the apoplast as it diffuses into a xylem tracheid or
pores. Because each plasmodesma is partly occluded by the vessel element. Again, the Casparian strip prevents the ion
desmotubule and associated proteins (see Chapter 1), the from diffusing back out of the root through the apoplast.
movement of large molecules such as proteins through the The presence of the Casparian strip allows the plant to
plasmodesmata requires special mechanisms (Ghoshroy et maintain a higher ionic concentration in the xylem than
al. 1997). Ions, on the other hand, appear to move from cell exists in the soil water surrounding the roots.
to cell through the entire plant by simple diffusion through
the symplast (see Chapter 4). Xylem Parenchyma Cells Participate in Xylem
Loading
Ions Moving through the Root Cross Both Once ions have been taken up into the symplast of the root
Symplastic and Apoplastic Spaces at the epidermis or cortex, they must be loaded into the tra-
Ion absorption by the roots (see Chapter 5) is more pro- cheids or vessel elements of the stele to be translocated to
nounced in the root hair zone than in the meristem and the shoot. The stele consists of dead tracheary elements and
106 Chapter 6
the living xylem parenchyma. Because the xylem tracheary NO3– are all taken up actively by the epidermal and corti-
elements are dead cells, they lack cytoplasmic continuity cal cells and are maintained in the xylem against a gradi-
with surrounding xylem parenchyma. To enter the tra- ent of electrochemical potential when compared with the
cheary elements, the ions must exit the symplast by cross- external medium (Lüttge and Higinbotham 1979). How-
ing a plasma membrane a second time. ever, none of these ions is at a higher electrochemical
The process whereby ions exit the symplast and enter potential in the xylem than in the cortex or living portions
the conducting cells of the xylem is called xylem loading. of the stele. Therefore, the final movement of ions into the
The mechanism of xylem loading has long baffled scien- xylem could be due to passive diffusion.
tists. Ions could enter the tracheids and vessel elements of However, other observations have led to the view that
the xylem by simple passive diffusion. In this case, the this final step of xylem loading may also involve active
movement of ions from the root surface to the xylem processes within the stele (Lüttge and Higinbotham 1979).
would take only a single step requiring metabolic energy. With the type of apparatus shown in Figure 6.19, it is pos-
The site of this single-step, energy-dependent uptake sible to make simultaneous measurements of ion uptake
would be the plasma membrane surfaces of the root epi- into the epidermal or cortical cytoplasm and of ion loading
dermal, cortical, or endodermal cells. According to the pas- into the xylem.
sive-diffusion model, ions move passively into the stele via By using treatments with inhibitors and plant hormones,
the symplast down a gradient of electrochemical potential, investigators have shown that ion uptake by the cortex and
and then leak out of the living cells of the stele (possibly ion loading into the xylem operate independently. For
because of lower oxygen availability in the interior of the example, treatment with the protein synthesis inhibitor
root) into the nonliving conducting cells of the xylem. cycloheximide or with the cytokinin benzyladenine inhibits
Support for the passive-diffusion model was provided xylem loading without affecting uptake by the cortex. This
by use of ion-specific microelectrodes to measure the elec- result indicates that efflux from the stelar cells is regulated
trochemical potentials of various ions across maize roots independently from uptake by the cortical cells.
(Figure 6.18) (Dunlop and Bowling 1971). Data from this Recent biochemical studies have supported a role for
and other studies indicate that K+, Cl–, Na+, SO42–, and the xylem parenchyma cells in xylem loading. The plasma
High
Chloride (Cl–)
Electrochemical
potential
Potassium (K+)
Low
Outside Xylem Xylem
Epidermis Cortex Endodermis parenchyma tracheary
solution
Stele
Casparian strip
FIGURE 6.18 Diagram showing electrochemical potentials chemical potential for both K+ and Cl– between the bathing
of K+ and Cl– across a maize root. To determine the electro- medium and the epidermis indicates that ions are taken up
chemical potentials, the root was bathed in a solution con- into the root by an active transport process. In contrast, the
taining 1 mM KCl and 0.1 mM CaCl2. A reference electrode potentials decrease at the xylem vessels, suggesting that
was positioned in the bathing solution, and an ion-sensitive ions are transported into the xylem by passive diffusion
measuring electrode was inserted in different cells of the down the gradient of electrochemical potential. (After
root. The horizontal axis shows the different tissues found Dunlop and Bowling 1971.)
in a root cross section. The substantial increase in electro-
Solute Transport 107
Compartment A Compartment B FIGURE 6.19 We can measure the relationship between ion
uptake into the root and xylem loading by placing a root seg-
ment across two compartments and adding a radioactive tracer
to one of them (in this case compartment A). The rate of disap-
Root pearance of the tracer from compartment A gives a measure of
segment ion uptake, and the rate of appearance in compartment B pro-
vides a measurement of xylem loading. (From Lüttge and
Radioactive Higinbotham 1979.)
tracer added