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236
Plasma membrane transport in context — making sense out
of complexity
Frans JM Maathuis* and Dale Sanders
Major advances in our understanding of the transport of
inorganic nutrient ions across plant plasma membranes have
emerged from recent studies on the control of the dominant
H+-pumping ATPase and from identification of a range of
new transporters for divalent cations, potassium, phosphate
and nitrate. In many cases, multiple transporter isoforms
have been described. An appreciation of the physiological
roles of these transporters demands combined genetic and
physiological approaches, which, in the case of an outward
rectifying K+ channel, have already been used to yield an
intriguing insight into root-mediated K+ release into the
xylem. In this review we attempt to place some of those
developments in a physiological context.
This simplistic picture for cation and anion transport —
although broadly correct — requires refinement. When
present at very low concentrations, transport of cations
such as K+ will require energisation by both components of
the PMF [4]. In such cases transport requires the operation
of more than one class of transport system. Furthermore,
many of the proteins which catalyse transport for a given
ion are expressed as a variety of isoforms. These findings
demand that the significance of multiple pathways for ion
transport be considered in a physiological context. New
insights are also emerging into the mechanisms of control
of the activities of H+-pumping ATPases, which underlie
plasma membrane energisation.
Addresses
The Plant Laboratory, Department of Biology, University of York,
PO Box 373, York YO1 5YW, UK
*e-mail: [email protected]
Important families of transporters for ammonium and sulphate have been identified some years ago and have been
discussed previously [5]. In this review we consider the
properties of recently-identified transporters for a number
of nutrient ions, including divalent cations, K+, Pi and
nitrate. Their physiological roles are considered in relation
to their expression patterns and, where studied, their
kinetic properties.
Current Opinion in Plant Biology 1999, 2:236–243
http://biomednet.com/elecref/1369526600200236
© Elsevier Science Ltd ISSN 1369-5266
Abbreviations
HAK
high affinity K+ transporter
KIRC
K+ selective inward rectifying channel
KORC K+ selective outward rectifying channel
LCT
low affinity cation transporter
PMF
protonmotive force
ZIP
ZRT, IRP-like protein
Regulation of H+ transport
Plant growth depends on the acquisition and appropriate
partitioning of inorganic solutes from the soil. The principal inorganic nutrients are potassium, nitrogen,
phosphate and sulphate [1]. The latter three nutrients
are metabolised and incorporated into organic compounds, while K+ plays a key role in generating osmotic
pressure and in compensating net negative charge of
cytosolic constituents [2]. Other essential nutrients,
which are absorbed to a lesser extent, include divalent
cations required as enzyme cofactors (iron, zinc, copper)
or for signal transduction (calcium).
The H+-ATPase reaction cycle includes a phosphorylated intermediate state. Both the phosphorylation and
ATP-binding domains are conserved. The H+-ATPase
exhibits a long hydrophilic extension around the carboxyl-terminus (Figure 1). Truncation of the
carboxyl-terminus elicits constitutive activity of the
enzyme, indicating that the carboxyl-terminus is a regulatory domain [6]. It has also been known for many years
that activity of the H+-ATPase is increased by the fungal
toxin fusicoccin [7], and that one potential site of interaction of fusicoccin is with 14-3-3 proteins [8]. 14-3-3
proteins are ubiquitous in all eukaryotic organisms where
they play essential roles in cell signalling, possibly by
modulating protein phosphorylation. Only recently, however, has the mode of action of fusicoccin on the
H+-ATPase been explained in a molecular context, that
has general relevance to the control of H+ pumping at
the plasma membrane.
Absorption of all nutrients is energised, ultimately, by proton pumps [3]. H+-ATPases are ubiquitous in plant plasma
membranes and generate an electrochemical potential difference for H+ (i.e. protonmotive force [PMF]). The
electrical component of the PMF — the membrane potential — is typically of the order –150 mV, and tends to drive
cation uptake requiring only the presence of a pore, or
channel. By contrast, accumulation of anionic nutrients
requires additional energization, which is provided by coupling their transport to the re-uptake of H+.
Co-purification of an active H+-ATPase and a 14-3-3 protein from fusicoccin-treated tissue, but not from control
tissue, indicates that direct interaction between the H+ATPase and 14-3-3 proteins is mediated by fusicoccin
[9•,10•,11]. Furthermore, cleavage of a 10 kDa fragment
from the carboxy-terminal of the H+-ATPase abolished
interaction with the 14-3-3 protein, reinforcing the
notion that fusicoccin-induced stimulation is related to
relief of auto-inhibition by the carboxy-terminal domain
[9•]. Expression in yeast of one isoform of the H+-
Introduction
Plasma membrane transport in context Maathuis and Sanders
237
Figure 1
The modulation of H+-ATPase activity by
fusicoccin (FC) and 14-3-3 proteins. The
ATPase activity shifts from low to high after
binding of both FC plus 14-3-3 to the
carboxyl-terminus (Ct) of the pump. Neither
the ATPase nor the 14-3-3 protein is capable
of FC binding on its own (see [3]).
Low-activity state
High-activity state
FC
Ct
14-3-3
Current Opinion in Plant Biology
ATPase from Arabidopsis thaliana, AHA2, has provided
further evidence for functional interactions between the
enzyme and 14-3-3 proteins [12••]. Thus, fusicoccin
binding activity was conferred by the carboxy-terminal
domain of AHA2, but only in the presence of 14-3-3 proteins. The fusicoccin binding site appears to be shared
between the H+-ATPase and the 14-3-3, although
whether an endogenous small molecule fulfils this function in planta is not known.
Emerging classes of divalent cation transporter
The molecular identity of plant uptake systems for divalent
cations has, until recently, been a mystery. Several developments in the past few years have, however, resulted in the
characterisation of divalent cation transporters, predominantly via functional complementation of yeast mutants.
An iron-regulated metal transporter gene (IRT1), encoding
a protein with eight putative transmembrane spans, has
Figure 2
Genes that encode transport systems in the
plant plasma membrane and their putative
functions as discussed in the text. CDPK;
calcium/calmodulin domain protein kinase.
K+
Xylem
Ca2+
Na+/K+
Na+
LCT1
CDPK
Ca2+
Root apoplast
Cd2+/
Ca2+?
KAT
H+?K+
nH+?Pi
KUP
HAK
AtKT
APT
PHT
LePT
MePT
H+?
NO3–
Guard cell
NRT1
NRT2
HKT
Vacuole
SKOR
K+
ATP
Ca2+
Root symplast
ADP
ZIP
AHA
ATP
ADP
H+
Zn2+
Current Opinion in Plant Biology
238
Physiology and metabolism
Table 1
Gene products involved in the uptake and translocation of K+ in plants.
Protein
Species
Type
Expression
Inhibitors
Function
Reference
AKT1
A. thaliana
Channel
Root cortex
Cs+/TEA/Ba2+
Low and high
affinity uptake
[18••]
SKOR
A. thaliana
Channel
Root pericycle
Xylem parenchyma
Translocation
to shoot
[25••]
HvHAK1–2
Barley
Wheat
Rice
A. thaliana
Carrier
Root
Na+/NH4+
High affinity uptake
(Km of 27 µM)
[19••]
AtKUP1–4
(AtKT1–2)
A. thaliana
Carrier
Flower/leaf
Root/stem
Cs+
High affinity uptake
(Km of 22 µM) with
low affinity component
[21•]
AtKUP1
A. thaliana
Carrier
Root
Cs+/Ba2+
Dual affinity uptake
(Kms of 44 µM and 11mM)
[20•]
Wheat
Barley
Carrier
Root
High affinity uptake
(Km of 3 µM), low
affinity Na+ uptake
Na+ uptake
[22–24]
HKT1
Rice
been identified in Arabidopsis [13]. Yeast complementation
experiments suggest that IRT1 is involved in Fe2+ transport. Expression is most marked in roots, and is induced by
iron deficiency. The IRT1 gene is a member of a family
which includes Zn2+ transporters encoded by the ZRT
genes of yeast, known as the ZIP (for ZRT, IRP-like protein) family (Figure 2).
In the case of Fe, transporter activity in delivering the ion to
the root cell is often supplemented by secretion of chelators
which mobilise Fe3+ from the soil. Further insights into the
mechanism of Fe uptake have emerged from the recent
identification of a membrane-bound ferric-chelate reductase
in Arabidopsis ( see Note added in proof). At the root surface,
Fe3+ → Fe2+ reduction reduces affinity for the chelator,
thereby increasing the concentration of Fe2+ for subsequent
uptake. The FRO2 gene was identified on the basis of
homology with human and yeast plasma membrane enzymes
that transfer electrons from FADH2 to an acceptor on the
opposing side of the membrane via two intramembrane haem
groups. FRO2 transcripts accumulate in roots in response to
Fe deficiency. Furthermore, FRO2 complements mutants
which are defective in ferric chelate reductase activity. The
deduced structure of FRO2 contains a cytosolic binding site
for FAD and conserved intramembrane His residues thought
to be involved in coordination of the two haems.
In complementation studies using yeast zrt mutants, four
genes (ZIP1–4), which encode transporters involved in
Zn2+ uptake, were identified in Arabidopsis [14••]. Each of
these transporters is predicted to possess 7–9 transmembrane spans, and expression of two (ZIP1 and ZIP3) is
preferential in root tissue and enhanced by Zn2+ starvation.
These findings suggest that ZIP1 and ZIP3 are involved in
Zn2+ uptake from the soil. The differential substrate specificity and expression patterns of ZIP isoforms might point
to their discrete physiological roles. Competition experiments with other divalent cations also indicate that ZIP1
and ZIP3 are distinctly Zn2–-specific, while ZIP2 exhibits
equal or greater affinity for Cu2+ and Cd2+.
Sequence analysis of ZIP family members demonstrates a
degree of conservation in putative transmembrane spans
IV, V and VI, possibly associated with an intramembrane
metal binding site [15•]. Furthermore, in the majority of
members, a possible heavy metal binding site has been
identified in the extramembrane loop between transmembrane spans III and IV. This conserved sequence has the
form HXHXH, a motif found in another, otherwise unrelated family of heavy metal transporters from bacteria.
A completely different type of transporter involved in
cation uptake has been identified in wheat roots and
shoots. The LCT1 (for low affinity cation transporter) gene
was originally identified by virtue of its ability to restore
growth of a K+ uptake-deficient yeast in low millimolar K+
concentrations [16]. LCT1 was originally shown to transport K+ and Na+ in yeast; however, further work
demonstrated an ability also to transport Ca2+ and Cd2+
[17••]. Although the physiological function of LCT1 is
unknown, this is the first transport system identified as a
candidate for mediating Ca2+ uptake by plant cells.
Our knowledge of the pathways for divalent cation transport
across the plasma membrane therefore remains fragmentary,
yet, significant advances can be expected in the coming
years, not only from complementation screens, but also from
bioinformatic approaches to genome sequences.
Plasma membrane transport in context Maathuis and Sanders
239
Table 2
Gene products involved in the uptake of phosphate in plants.
Protein
Species
Expression
Transport Km
Reference
APT1–2 (APT1–4)
A. thaliana
Root/low level in leaf
–
[33,35•]
PHT1
A. thaliana
Root
3 µM
[34]
Potato
Root/tuber/leaf flower
280 and 130 µM
[31•]
Tomato
Root/mature leaf
31 µM
[30•]
M. trunculata
Root
192 µM
[32•]
StPT1–2
LePT1
MtPT1–2
Potassium transport
To maintain the essential roles that K+ plays in cellular
homeostasis requires sophisticated means to move K+ at
key locations throughout the plant, which include the
soil/root interface, the xylem, and cells involved in movement, for example in guard cells, which are responsible for
the opening and closing of stomatas.
K+ transport at the soil/root interface
The process of K+ uptake in plant roots has been a major
focus of plant physiology. Conventionally, K+ accumulation
at various ambient K+ concentrations is perceived as occurring at the root cell plasma membrane through two or more
independent influx systems with respectively high and low
affinities. Traditionally, low affinity K+ uptake is believed
to be mediated by K+ selective ion channels, a notion supported by both patch clamp characterisation of root K+
channels and yeast complementation studies [2].
Thermodynamic constraints require energisation of high
affinity K+ uptake via carriers [4].
The convenient notion of a channel and carrier mediated
low and high affinity K+ uptake is rapidly crumbling in the
face of recent data. Functional analysis of the role of AKT1
(a K+ selective inward rectifying channel or KIRC) in planta has been advanced [18••] by identifying an AKT1 null
mutant (akt1-1) from an Arabidopsis T-DNA mutagenised
population. Surprisingly, differences in growth between
wild type and akt1-1 were apparent only in the presence of
millimolar ammonium and with K+ levels of >1 mM. In the
presence of ammonium, Rb+ uptake from external solutions was reduced, but progressively so at lower Rb+
concentrations. A reduced Rb+ influx in the mutant also
occurred in the absence of ammonium conditions where no
phenotype was observed. The combined results of this
study indicate that, in the presence of ammonium, the
AKT1 channel plays a role in high affinity K+ uptake.
They also indicate that AKT1 may not be the predominant low affinity K+ influx pathway in A. thaliana roots, as
at 1 mM external Rb+ concentrations the mutant shows
around 70% of the wild-type uptake.
With a sufficiently negative membrane potential, the
influx of K+, even from lower micromolar concentrations,
can proceed ‘down hill’ and this obviates the requirement
for an energised carrier mechanism. Nevertheless, a large
number of carrier type K+ transporters has now been
identified (Table 1, Figure 2). In barley, HvHAK was
cloned [19••] using degenerate primers for conserved
regions of the Schwanniomyces HAK (high affinity K+)
transporter. Complementation of K+ transport deficient
yeast restored high affinity Rb+ uptake, which, in agreement with the observations of Hirsch et al. [18••], was
sensitive to ammonium. The HvHAK1 protein has 12
putative membrane spanning regions and is a member of
a large conserved family of transporters that are highly K+
selective and probably function as H+ coupled systems
[19••]. The barley member of this family shows homology to HAK/KUP transporters found in bacteria
(Escherichia coli), fungi (Schwanniomyces occidentalis),
plants (Lyphopyrum, wheat, rice and Arabidopsis) and animals (Homo sapiens). HvHAK1 transcript is exclusively
found in roots and highly induced by K+ starvation, which
is in agreement with the frequently observed induction of
high affinity K+ uptake in intact plants.
By searching for HAK/KUP homologues in A. thaliana
EST databases, several groups isolated a number of
HAK/KUP isoforms (AtKUP 1–4). The Rb+ and K+ transport capacity of AtKUP1 was analysed in homologous
and heterologous expression systems [20•,21•]. In both
expression systems AtKUP1 mediated high affinity Rb+
transport with a micromolar Km indicating a role in high
affinity K+ transport in vivo. In addition to high affinity
K+ transport, AtKUP1 expressing yeast cells showed a
rise in Rb+ uptake from millimolar concentrations [20•].
The latter suggests that HAK/KUP type proteins may
also contribute to low affinity K+ transport. Expression
patterns of AtKUP1 differ in various reports (Table 1) and
expression of at least one isoform (AtKUP3) is up regulated upon K+-starvation [21•].
A class of putative high affinity K+ transporters, not related to HAK/KUP, is provided by HKT1 type mechanisms
[22]. Originally cloned from wheat, HKT1 catalyses
K+:Na+ (µM ambient Na+) or Na+:Na+ (mM ambient Na+)
symport. Homologues of HKT1 have been found in barley [23], rice [24] and A. thaliana [22] and in both barley
and wheat mRNA levels increase in K+ starvation conditions [23]. The physiological relevance of HKT1 remains
240
Physiology and metabolism
Figure 3
Model according to Doyle et al. 1998 [44] for
the permeation of K+ ions through an inward
rectifying K+ channel with a pore region
similar to that of the Shaker family K+
channels. Ions traverse a narrow pore within
the channel, which forms the selectivity filter
(SF) and can hold two ions simultaneously.
The large aqueous cavity helps to stabilise
cation(s) in the middle of the membrane by
the dipole action of the depicted helices.
SF
–
–
Current Opinion in Plant Biology
to be clarified but may be in providing a pathway for Na+
entry into the plant [24].
K+ release into the xylem
SKOR [25••] is an Arabidopsis K+ selective channel and a
member of the Shaker gene family (Figure 3). Shaker type
channels are K+ selective, voltage gated outward rectifying
channels that were first identified in Drosophila shaker
mutants. In spite of the large degree of homology to KAT
and AKT inward rectifying channels, SKOR mediates K+
efflux. How such similar primary protein structures result
in K+ channels with contrasting voltage sensitivity remains
to be explained. Expression patterns and gene disruption
mutants imply a clearly defined function for SKOR in
xylem loading of K+: GUS constructs with the SKOR promoter show expression restricted to the root pericycle and
xylem parenchyma cells. Disruption of the SKOR gene did
not affect root K+ levels but caused a decrease in xylem sap
K+ and a reduction in shoot K+ contents, consistent with a
function in K+ release into the xylem. Exposure of plants
to the stress hormone ABA, which is believed to play a role
in ion translocation to the shoot, resulted in a rapid
decrease of SKOR transcript.
K+ transport in guard cells
The first plant K+ channels were identified in guard cells
which control stomatal aperture by osmotic swelling and
shrinking caused by the movement of large amounts of
K+. Via the membrane voltage, either inward or outward
rectifying channels are activated to allow a sustained inward
or outward flux of K+ for stomatal opening or closing,
respectively. A variety of stimuli such as ABA, CO2 and
oxidative stress promote stomatal closure, probably via a
raise in cytosolic Ca2+. A direct target for cytosolic Ca2+
Figure 4
NH2
COOH
Current Opinion in Plant Biology
Generalised model for the topology of high
affinity phosphate carriers. Transmembrane
spans show an internal repeat and are
arranged in a typical 6+6 structure
characteristic of many carriers belonging to
the major facilitator superfamily. The large
cytoplasmic loop contains a putative
phosphorylation site for kinase C.
Plasma membrane transport in context Maathuis and Sanders
has now been described in Vicia faba guard cells [26] as a
Ca2+ dependent protein kinase that phosphorylates and
down regulates KAT1, the predominant KIRC in guard
cell plasma membranes.
241
result from the use of Saccharomyces as expression system
where the interaction of several proteins is necessary for the
proper functioning of high affinity phosphate transport [36].
Cytosolic Ca2+ also affects the activity of a newly identified
K+ selective outward rectifying channel (KORC) [27]. In
contrast to previously described KORCs that show sustained currents, this particular type of KORC rapidly
inactivates. The physiological role of this channel remains
to be elucidated, but may be in the transduction of signals
during stomatal closing. Additional potential modulators of
stomatal aperture that act on K+ channels are agents that
affect the polymerisation of actin filaments [28] and the
osmolarity of solutions facing the membrane [29].
The relative contribution of these gene products to the
overall uptake of phosphate by plants will be affected by
the formation of mycorrhizae, which can drastically
improve plant phosphate nutrition. The presence of mycorrhizae will shift phosphate uptake to GvPT-type
transporters at the soil/fungus interface and can reduce the
amount taken up by the plant root to almost nil. The
mechanism of phosphate transfer from fungus to root
remains unknown but probably involves separate gene
products as was shown for Medicago where MtPT transcript
was significantly reduced after mycorrhiza formation [32•].
Phosphate uptake in plant roots
Nitrate uptake
Phosphate is an essential macronutrient and plant cells control cytosolic phosphate within narrow limits by balancing
import and export. Rates of phosphate uptake depend on
growth demand and phosphate supply, the latter frequently being inadequate due to the low mobility of phosphate
in the soil. At the prevailing soil pH, phosphate is most
readily taken up by plants as H2PO4–. External concentrations of this nutrient are typically a few micromolar in
physiological conditions, whereas cytosolic levels are a
1000-fold higher. Phosphate transport shows a pronounced
pH optimum, is highly sensitive to uncouplers [30•] and is
generally assumed to occur via H+ coupled symport.
Nitrate uptake from the soil has long been suspected,
from kinetic studies, to involve an array of different transport systems. Thus, constitutive high affinity and
non-saturable kinetic phases exist in roots along with a
high affinity phase, which is induced by nitrate [38]. The
properties of the transport systems that underlie this
kinetic complexity are beginning to be elucidated with
candidate genes falling into two families.
Several genes encoding high affinity phosphate transporters have been cloned (Table 2; [30•–32•,33,34,35•]).
The proteins encoded by these genes are around 60 kD,
and show high levels of homology to each other and to the
high affinity phosphate transporters of yeast PHO84 [36]
and mycorrhizal fungi GvPT [37]. APT1 and APT2 [35•] for
example, are both expressed in Arabidopsis root tissue and
share 99% identity in their coding regions; however, their
promoter regions are very different, pointing to cell-typespecific or development-specific expression for either gene.
Hydrophobicity plots show a common structure of
12 transmembrane spans that contain an internal repeat of
6 + 6 transmembrane spans (Figure 4) with a large central
hydrophobic region protruding into the cytosol that carries
a highly conserved putative kinase phosphorylation site. In
some cases transcript levels were shown to be sensitive to
the phosphate status of the plant (e.g. for APT1, APT2)
whereas other genes appear to be constitutively expressed
(e.g. for StPT2).
The functional analysis of LePT1 from tomato [30•] and of
MtPT1 from Medicago [32•] was carried out in the yeast
pho84 strain. In both cases, yeast growth was restored on
micromolar phosphate and transport assays confirmed the
restoration of phosphate uptake with an apparent Km of 31
µM for LePT1 and 192 µM for MtPT1. Both Km values are
much higher than those established in intact plants and may
The first family of nitrate transporters is identified by a
consensus motif (FYXXINXGSL), characteristic of the
PTR family of peptide transporters present in yeast,
plants and mammals. At least two members of this family are present in Arabidopsis: NRT1 (or CHL1) and
NRT3 (or NLT3) [38]. NRT1 expression is inducible by
nitrate, whereas that of NRT3 appears to be constitutive.
Until recently both transport systems were thought to be
involved only in low affinity transport. Careful analysis
of one chl1 mutant line containing an active transposable
element in CHL1, however, revealed defects in both low
and high affinity transport [39••]. The extent to which
CHL1 participates in high affinity transport depends
critically on growth conditions: the presence of ammonium ions results in a marked contribution of CHL1 to
high affinity uptake, whereas in the absence of ammonium no contribution is apparent. CHL1, therefore,
appears to behave as a dual affinity transporter, a phenomenon that can be explained by random binding of H+
and nitrate to the carrier [40].
A CHL1 homologue from Brassica napus has been functionally characterised in oocytes with respect to substrate
specificity [41••]. Generally, transport carriers exhibit a
high degree of substrate specificity and it was, therefore,
surprising to find that CHL1 not only translocates nitrate,
but also histidine and to a lesser extent, other basic amino
acids. The physiological significance of this observation
is not known but could relate to delivery of amino acids
to growing cells. Resolution of expression patterns at a
cellular level could help resolve this issue; it is already
known that two putative nitrate transporters in the PTR
242
Physiology and metabolism
family exhibit differential expression patterns in tomato
roots [42].
Members of the second nitrate transporter family (NRT2)
are nitrate inducible, belong to the major facilitator superfamily and were identified originally on the basis of
homologies to fungal and algal nitrate transporters [38].
Confirmation of a role in nitrate transport awaits their functional expression or the identification of mutants in these
genes. The high degree of correlation between expression
levels of the tobacco transporter NRT2 with high affinity
nitrate transport activity, however, provides evidence for a
function in inducible, high affinity nitrate transport [43•].
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
1.
Marschner H: Mineral Nutrition of Higher Plants. London: Academic
Press; 1995
2.
Maathuis FJM, Sanders D: Mechanisms of potassium absorption by
higher plant roots. Physiol Plant 1996, 96:158-168.
3.
Palmgren MG, Fuglsang AT, Jahn T: Deciphering the role of 14-3-3proteins. Exp Biol On line 1998, 3:1-20.
4.
Maathuis FJM, Sanders D: Energization of potassium uptake in
Arabidopsis thaliana. Planta 1993, 191:302-307.
5.
Assmann SM, Haubrick LL: Transport proteins of the plant plasma
membrane. Curr Opin Cell Biol 1996, 8:458-467.
6.
Palmgren MG, Larsson C, Sommarin M: Proteolytic activation of the
plant plasma-membrane H+-ATPase by removal of a terminal
segment. J Biol Chem 1990, 265:13423-13426.
7.
Radice M, Scacchi A, Pesci P, Beffagna N, Marre MT: Comparativeanalysis of the effects of fusicoccin (fc) and of its derivative
dideacetylfusicoccin (daf) on maize leaves and roots. Physiol Plant
1981, 51:215-221.
8.
Korthout HAAJ, deBoer AH: A fusicoccin binding-protein belongs
to the family of 14-3-3-brain protein homologs. Plant Cell 1994,
6:1681-1692.
Conclusions and prospects
The number of transporters identified at a molecular
level has increased dramatically over the past years. For
any given nutrient, an array of potential transport pathways is present at the plasma membrane, even within a
single species.
At one level, this complexity reflects the presence of isoforms that are often expressed on a tissue- or cell
type-specific basis. Isoforms enable transport to be controlled at a transcriptional level in response to
developmental or environmental signals. At a second
level, different classes of transporter are apparent for ions
like K+ and nitrate which may reflect the wide range of
nutrient concentrations to which plants are exposed.
Although the complexity of multiple transport pathways
will doubtless increase as genomic information expands,
we can expect marked advances in our understanding of
the functional attributes of transporters with the combination of reverse genetic and physiological approaches over
the coming years.
Jahn T, Fuglsang AT, Olsson A, Bruntrup IM, Collinge DB,
Volkmann D, Sommarin M, Palmgren MG, Larsson C: The 14-3-3
protein interacts directly with the c-terminal region of the plant
plasma membrane H+-ATPase. Plant Cell 1997, 9:1805-1814.
In this study, detergent-solubilised plasma membrane H+-ATPase isolated
from fusicoccin-treated maize was copurified with the 14-3-3 protein, yielding H+-ATPase in an activated state. In the absence of fusicoccin treatment,
H+-ATPase was recovered in a nonactivated form.
Considerable effort and expense is required for the inorganic fertilisation of many agricultural systems. Many
such systems are, indeed, over-fertilised, with devastating
consequences for water courses which become eutrophic.
Identification of those transporters which are pivotal in
the uptake of inorganic nutrients from the soil and the
subsequent redistribution of nutrients around the plant
offers the exciting possibility of engineering more efficient strategies for fertiliser application. Such strategies
should focus not only on the mechanisms for solute
uptake, involving, for example, the affinity of the transporter for the ion. In addition the clear implication of the
presence of multiple isoforms is that transcriptional and/or
post-translational controls might differ, and elucidating
the pathways leading to regulation of transporter activity
can be predicted to become a new and major goal of studies in nutrient acquisition.
12. Baunsgaard L, Fuglsang AT, Jahn T, Korthout HAAJ, deBoer AH,
•• Palmgren MG: The 14-3-3 proteins associate with the plant plasma
membrane H+-ATPase to generate a fusicoccin binding complex
and a fusicoccin responsive system. Plant J 1998, 13:661-671.
By testing the fusicoccin binding activity of yeast membranes, the carboxy-terminal regulatory domain of AHA2 was found to be part of a functional fusicoccin
receptor, a component of which was the 14-3-3 protein. ATP hydrolytic activity of
AHA2 expressed in yeast internal membranes was activated by all tested isoforms
of the 14-3-3 protein of yeast and Arabidopsis, but only in the presence of fusicoccin.
9.
•
10. Oecking C, Piotrowski M, Hagemeier J, Hagemann K: Topology and
•
target interaction of the fusicoccin-binding 14-3-3 homologs of
Commelina communis. Plant J 1997, 12:441-453.
Experiments carried out with sealed plasma membrane vesicles show that
the fusicoccin-binding site faces the cytoplasmic surface of the membrane.
Stabilisation of the labile ATPase–14-3-3 complex in plasma membranes
could be achieved by fusicoccin treatment in vivo or in vitro. The carboxylterminus probably represents the binding domain for 14-3-3 homologues.
11. Oecking C, Hagemann K: Association of 14-3-3 proteins with the
C-terminal antoinhibitory domain of the plant plasma-membrane
H+-ATPase generates a fusicoccin-binding complex. Planta 1999,
207:480-482.
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Report on the identification of ZIP genes from Arabidopsis as encoding Zn2+
transporters. Expression in yeast reveals high specificity of two members of the
family for Zn2+, and transcript levels are upregulated in roots on Zn2+ starvation.
The plant ZIP transporters are related to metal transporters found in many other
eukaryotes, including yeast, protozoa and — more distantly — humans.
Note added in proof
15. Eng BH, Guerinot ML, Eide D, Saier MH: Sequence analyses and
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phylogenetic characterization of the ZIP family of metal ion
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Plasma membrane transport in context Maathuis and Sanders
Clemens S, Antosiewicz DM, Ward JM, Schachtman DP: The
plant cDNA LCT1 mediates the uptake of calcium and cadmium in
yeast. Proc Natl Acad Sci USA 1998, 95:12043-12048.
Expression of LCT1 in yeast renders growth of yeast more sensitive to Cd2+.
Ion flux assays showed an increase in Cd2+ uptake. Growth assays also
demonstrate a sensitivity of LCT1-expressing yeast cells to extracellular millimolar Ca2+ concentrations. It is concluded that LCT1 can mediate uptake
of Ca2+ and Cd2+ in yeast and possibly also in plants.
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••
18. Hirsch RE, Lewis BD, Spalding EP, Sussman MR: A role for the AKT1
•• potassium channel in plant nutrition. Science 1998, 280:918-921.
Reverse genetic screening identified an Arabidopsis thaliana mutant in
which the AKT1 channel gene was disrupted. Roots of this mutant lacked
inward-rectifying potassium channels and displayed reduced potassium
(rubidium-86) uptake in the presence of ammonium.
19. Santa-Maria G, Rubio F, Dubcovsky J, Rodriguez-Navarro A: The
•• HAK1 gene of barley is a member of a large gene family and
encodes a high-affinity potassium transporter. Plant Cell 1997,
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K+ transporter that is expressed in root cells and which shows a high degree
of homology to bacterial high affinity K+ transporters.
20. Fu H, Luan S: AtKUP1: a dual affinity K+ transporter from
•
Arabidopsis. Plant Cell 1998, 10:63-67.
Characterisation of AtKUP3 — a K+ transporter — in yeast. When expressed
in yeast, AtKUP3 functions in both high-and low-affinity uptake.
21. Kim EJ, Kwak JM, Uozumi N, Schroeder JI: AtKUP1: an Arabidopsis
•
gene encoding high-affinity potassium transport activity. Plant
Cell 1998, 10:51-62.
The identification of AtKUP from Arabidopsis thaliana via homology screening
of non-plant Kup and of HAK1 potassium transporters from Escherichia coli
and Schwanniomyces occidentalis. AtKUP1 and AtKUP2 are able to complement a potassium transport deficient E. coli triple mutant. Kinetic characterisation and expression patterns are reported. Through sequence analysis a family
specific signature sequence and metol-binding domains are described.
22. Schachtman DP, Schroeder JI: Structure and transport mechanism
of a high-affinity potassium uptake transporter from higher
plants. Nature 1994, 370:655-658.
23. Wang TB, Gassmann W, Rubio F, Schroeder JI, Glass ADM: Rapid
upregulation of HKT1, a high-affinity potassium transporter gene,
in roots of barley and wheat following withdrawal of potassium.
Plant Physiol 1998, 118:651-659.
24. Golldack D, Kamasani UR, Quigley F, Bennett J, Bohnert HJ: Salt
stress-dependent expression of a HKT1-type high affinity
potassium transporter in rice. Plant Physiol 1997, 114:529
25. Gaymard F, Pilot G, Lacombe B, Bouchez D, Bruneau D, Boucherez J,
•• Michaux-Ferriere M, Thibaud J, Sentenac H: Identification and
disruption of a plant Shaker-like outward channel involved in K+
release into the xylem sap. Cell 1998, 94:647-655.
SKOR, a K+ channel identified in Arabidopsis, displays the typical hydrophobic
core of the Shaker channel superfamily. Expression in Xenopus oocytes identified SKOR as the first member of the Shaker family in plants to be outwardly
rectifying. SKOR expression is localised in root stelar tissues and evidence indicates that SKOR is involved in K+ release into the xylem sap toward the shoots.
26. Li J, Lee YR, Assmann SM: Guard cells possess a calcium-dependent
protein kinase that phosphorylates the KAT1 potassium channel.
Plant Physiol 1998, 116:785-795.
27.
Pei Z, Baizabal-Aguirre VM, Allen GJ, Schroeder JI: A transient
outward rectifying K+ channel current down-regulated by cytosolic
Ca2+ in Arabidopsis thaliana guard cells. Proc Natl Acad Sci USA
1998, 95:6548-6553.
28. Hwang J, Suh S, Yi H, Kim J, Lee Y: Actin filaments modulate both
stomatal opening and inward K+ channel activities in guard cells
of Vicia faba L. Plant Physiol 1997, 115:335-342.
29. Liu K, Luan S: Voltage-dependent K+ channels as a target of
osmosensing in guard cells. Plant Cell 1998, 10:1957-1970.
30. Daram P, Brunner S, Persson PL, Amrhein N, Bucher M: Functional
•
analysis and cell-specific expression of a phosphate transporter
from tomato. Planta 1998, 206:225-233.
Expression of LePT1, high affinity phosphate transporter from tomato, in a
phosphate-uptake-deficient yeast mutant. Phosphate transport shows a Km
of 31 µM and is highly dependent on the external pH. The work presents
molecular and biochemical evidence for distinct root cells playing an important role in phosphate acquisition at the root/soil interface.
31. Leggewie G, Wilmitzer L, Riesmeier JW: Two cDNAs from potato are
•
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identification of phosphate transporters from higher plants. Plant
Cell 1997, 9:381-392.
Description and isolation of two cDNAs, StPT1 and StPT2, from potato that
show homology to the phosphate/proton cotransporter from yeast.
32. Liu H, Trieu AT, Blaylock LA, Harrison MJ: Cloning and
•
characterization of two phosphate transporters from Medicago
trunculata roots: regulation in response to phosphate and
colonization by arbuscular mycorrhizal (AM) fungi. Mol
Plant–Microbe Interact 1998, 11:14-22.
Identification of two cDNA clones (MtPT1 and MtPT2) encoding phosphate
transporters from a mycorrhizal root cDNA library (Medicago truncatula/Glomus
versiforme). The cDNAs represent M. truncatula genes and the encoded proteins share identity with high-affinity phosphate transporters from Arabidopsis,
potato, yeast, Neurospora, and the arbuscular mycorrhizal fungus G. versiforme.
33. Lu YP, Zhen RG, Rea PA: AtPT4: a fourth member of the
Arabidopsis phosphate transporter gene family. Plant Physiol
1997, 114:747-751.
34. Mitsukawa N, Okumura S, Shirano Y, Sato S, Kato T, Harashima S,
Shibata D: Overexpression of an Arabidopsis thaliana high-affinity
phosphate transporter gene in tobacco cultured cells enhances
cell growth under phosphate-limited conditions. Proc Natl Acad
Sci USA 1997, 94:7098-7102.
35. Smith FW, Ealing PM, Dong B, Delhaize E: The cloning of two
•
Arabidopsis genes belonging to a phosphate transporter family.
Plant J 1997, 11:83-92.
Two genes, APT1 and APT2, with DNA sequences that exhibit significant
sequence identity to yeast and fungal H+/orthophosphate co-transporters,
isolated from Arabidopsis thaliana are described. Both are predominantly
expressed in root tissues and the level of expression is regulated by the
phosphorus status of the plant.
36. Yompakdee C, Ogawa N, Harashima S, Oshima Y: A putative
membrane protein, PHO88p, involved in inorganic phosphate
transport in Saccharomyces cerevisiae. Mol Gen Genetics 1996,
251:580-590.
37.
Harrison MJ, VanBuuren ML: A phosphate transporter from the
mycorrhizal fungus Glomus versiforme. Nature 1995, 378:626-629.
38. Crawford NM, Glass ADM: Molecular and physiological aspects of
nitrate uptake in plants. Trends Plant Sci 1998, 3:389-395.
39. Wang RC, Liu D, Crawford NM: The Arabidopsis CHL1 protein
•• plays a major role in high-affinity nitrate uptake. Proc Natl Acad
Sci USA 1998, 95:15134-15139.
A search performed to find high-affinity phosphate uptake mutants by using
chlorate selections on plants containing Tag1 transposable elements yielded a chlorate-resistant mutant with a Tag1 insertion in CHL1. Analysis
showed that chl1 mutants have reduced high-affinity uptake in addition to
reduced low-affinity uptake.
40. Sanders D: Generalized kinetic analysis of ion driven cotransport
systems: II. Random ligand binding as a simple explanation for
non-Michaelis kinetics. J Membr Biol 1986, 90:67-87.
41. Zhou JJ, Theodoulou FL, Muldin I, Ingemarsson B, Miller AJ: Cloning
•• and functional characterization of a Brassica napus transporter
that is able to transport nitrate and histidine. J Biol Chem 1998,
273:12017-12023.
A full-length cDNA for a membrane transporter was isolated from Brassica
napus by its sequence homology to a previously cloned Arabidopsis low
affinity nitrate transporter. The properties of the transporter are consistent
with a proton cotransport mechanism for nitrate. Histidine is also transported with an affinity was in the millimolar range.
42. Lauter FR, Ninnemann O, Bucher M, Riesmeier JW, Frommer WB:
Preferential expression of an ammonium transporter and of two
putative nitrate transporters in root hairs of tomato. Proc Natl
Acad Sci USA 1996, 93:8139-8144.
43. Krapp A, Fraisier V, Scheible WR, Quesada A, Gojon A, Stitt M,
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Caboche M, Daniel-Vedele F: Expression studies of NRT2:1NP, a
putative high-affinity nitrate transporter: evidence for its role in
nitrate uptake. Plant J 1998, 14:723-731.
Expression levels of the gene Nrt2Np, a putative high-affinity nitrate transporter
of Nicotiana plumbaginifolia, are studied under variable physiological conditions.
44. Doyle DA, Cabral JM, Pfeutzner RA, Kuo A, Gulbis JM, Cohen SL, Chait
BT, MacKinnon R: The structure of the potassium channel: molecular
basis of K+ conduction and selectivity. Science 1998, 280:69-77.
45. Robinson NJ, Proctor CM, Connolly EL, Guerinot ML: A ferric-chelate
•• reductase for ion uptake from soils. Nature 1998, 397:694-697.
Identification and characterisation of a gene involved in the reduction of ferric chelates in Arabidopsis roots. The gene product, FRO2, has a putative
binding site for FAD on the cytosolic side of the membrane, and coordination sites for two intramembrane haems which are thought to be involved in
reduction of Fe3+ on the external surface.
Plasma membrane transport in context — making sense out
of complexity
Frans JM Maathuis* and Dale Sanders
Major advances in our understanding of the transport of
inorganic nutrient ions across plant plasma membranes have
emerged from recent studies on the control of the dominant
H+-pumping ATPase and from identification of a range of
new transporters for divalent cations, potassium, phosphate
and nitrate. In many cases, multiple transporter isoforms
have been described. An appreciation of the physiological
roles of these transporters demands combined genetic and
physiological approaches, which, in the case of an outward
rectifying K+ channel, have already been used to yield an
intriguing insight into root-mediated K+ release into the
xylem. In this review we attempt to place some of those
developments in a physiological context.
This simplistic picture for cation and anion transport —
although broadly correct — requires refinement. When
present at very low concentrations, transport of cations
such as K+ will require energisation by both components of
the PMF [4]. In such cases transport requires the operation
of more than one class of transport system. Furthermore,
many of the proteins which catalyse transport for a given
ion are expressed as a variety of isoforms. These findings
demand that the significance of multiple pathways for ion
transport be considered in a physiological context. New
insights are also emerging into the mechanisms of control
of the activities of H+-pumping ATPases, which underlie
plasma membrane energisation.
Addresses
The Plant Laboratory, Department of Biology, University of York,
PO Box 373, York YO1 5YW, UK
*e-mail: [email protected]
Important families of transporters for ammonium and sulphate have been identified some years ago and have been
discussed previously [5]. In this review we consider the
properties of recently-identified transporters for a number
of nutrient ions, including divalent cations, K+, Pi and
nitrate. Their physiological roles are considered in relation
to their expression patterns and, where studied, their
kinetic properties.
Current Opinion in Plant Biology 1999, 2:236–243
http://biomednet.com/elecref/1369526600200236
© Elsevier Science Ltd ISSN 1369-5266
Abbreviations
HAK
high affinity K+ transporter
KIRC
K+ selective inward rectifying channel
KORC K+ selective outward rectifying channel
LCT
low affinity cation transporter
PMF
protonmotive force
ZIP
ZRT, IRP-like protein
Regulation of H+ transport
Plant growth depends on the acquisition and appropriate
partitioning of inorganic solutes from the soil. The principal inorganic nutrients are potassium, nitrogen,
phosphate and sulphate [1]. The latter three nutrients
are metabolised and incorporated into organic compounds, while K+ plays a key role in generating osmotic
pressure and in compensating net negative charge of
cytosolic constituents [2]. Other essential nutrients,
which are absorbed to a lesser extent, include divalent
cations required as enzyme cofactors (iron, zinc, copper)
or for signal transduction (calcium).
The H+-ATPase reaction cycle includes a phosphorylated intermediate state. Both the phosphorylation and
ATP-binding domains are conserved. The H+-ATPase
exhibits a long hydrophilic extension around the carboxyl-terminus (Figure 1). Truncation of the
carboxyl-terminus elicits constitutive activity of the
enzyme, indicating that the carboxyl-terminus is a regulatory domain [6]. It has also been known for many years
that activity of the H+-ATPase is increased by the fungal
toxin fusicoccin [7], and that one potential site of interaction of fusicoccin is with 14-3-3 proteins [8]. 14-3-3
proteins are ubiquitous in all eukaryotic organisms where
they play essential roles in cell signalling, possibly by
modulating protein phosphorylation. Only recently, however, has the mode of action of fusicoccin on the
H+-ATPase been explained in a molecular context, that
has general relevance to the control of H+ pumping at
the plasma membrane.
Absorption of all nutrients is energised, ultimately, by proton pumps [3]. H+-ATPases are ubiquitous in plant plasma
membranes and generate an electrochemical potential difference for H+ (i.e. protonmotive force [PMF]). The
electrical component of the PMF — the membrane potential — is typically of the order –150 mV, and tends to drive
cation uptake requiring only the presence of a pore, or
channel. By contrast, accumulation of anionic nutrients
requires additional energization, which is provided by coupling their transport to the re-uptake of H+.
Co-purification of an active H+-ATPase and a 14-3-3 protein from fusicoccin-treated tissue, but not from control
tissue, indicates that direct interaction between the H+ATPase and 14-3-3 proteins is mediated by fusicoccin
[9•,10•,11]. Furthermore, cleavage of a 10 kDa fragment
from the carboxy-terminal of the H+-ATPase abolished
interaction with the 14-3-3 protein, reinforcing the
notion that fusicoccin-induced stimulation is related to
relief of auto-inhibition by the carboxy-terminal domain
[9•]. Expression in yeast of one isoform of the H+-
Introduction
Plasma membrane transport in context Maathuis and Sanders
237
Figure 1
The modulation of H+-ATPase activity by
fusicoccin (FC) and 14-3-3 proteins. The
ATPase activity shifts from low to high after
binding of both FC plus 14-3-3 to the
carboxyl-terminus (Ct) of the pump. Neither
the ATPase nor the 14-3-3 protein is capable
of FC binding on its own (see [3]).
Low-activity state
High-activity state
FC
Ct
14-3-3
Current Opinion in Plant Biology
ATPase from Arabidopsis thaliana, AHA2, has provided
further evidence for functional interactions between the
enzyme and 14-3-3 proteins [12••]. Thus, fusicoccin
binding activity was conferred by the carboxy-terminal
domain of AHA2, but only in the presence of 14-3-3 proteins. The fusicoccin binding site appears to be shared
between the H+-ATPase and the 14-3-3, although
whether an endogenous small molecule fulfils this function in planta is not known.
Emerging classes of divalent cation transporter
The molecular identity of plant uptake systems for divalent
cations has, until recently, been a mystery. Several developments in the past few years have, however, resulted in the
characterisation of divalent cation transporters, predominantly via functional complementation of yeast mutants.
An iron-regulated metal transporter gene (IRT1), encoding
a protein with eight putative transmembrane spans, has
Figure 2
Genes that encode transport systems in the
plant plasma membrane and their putative
functions as discussed in the text. CDPK;
calcium/calmodulin domain protein kinase.
K+
Xylem
Ca2+
Na+/K+
Na+
LCT1
CDPK
Ca2+
Root apoplast
Cd2+/
Ca2+?
KAT
H+?K+
nH+?Pi
KUP
HAK
AtKT
APT
PHT
LePT
MePT
H+?
NO3–
Guard cell
NRT1
NRT2
HKT
Vacuole
SKOR
K+
ATP
Ca2+
Root symplast
ADP
ZIP
AHA
ATP
ADP
H+
Zn2+
Current Opinion in Plant Biology
238
Physiology and metabolism
Table 1
Gene products involved in the uptake and translocation of K+ in plants.
Protein
Species
Type
Expression
Inhibitors
Function
Reference
AKT1
A. thaliana
Channel
Root cortex
Cs+/TEA/Ba2+
Low and high
affinity uptake
[18••]
SKOR
A. thaliana
Channel
Root pericycle
Xylem parenchyma
Translocation
to shoot
[25••]
HvHAK1–2
Barley
Wheat
Rice
A. thaliana
Carrier
Root
Na+/NH4+
High affinity uptake
(Km of 27 µM)
[19••]
AtKUP1–4
(AtKT1–2)
A. thaliana
Carrier
Flower/leaf
Root/stem
Cs+
High affinity uptake
(Km of 22 µM) with
low affinity component
[21•]
AtKUP1
A. thaliana
Carrier
Root
Cs+/Ba2+
Dual affinity uptake
(Kms of 44 µM and 11mM)
[20•]
Wheat
Barley
Carrier
Root
High affinity uptake
(Km of 3 µM), low
affinity Na+ uptake
Na+ uptake
[22–24]
HKT1
Rice
been identified in Arabidopsis [13]. Yeast complementation
experiments suggest that IRT1 is involved in Fe2+ transport. Expression is most marked in roots, and is induced by
iron deficiency. The IRT1 gene is a member of a family
which includes Zn2+ transporters encoded by the ZRT
genes of yeast, known as the ZIP (for ZRT, IRP-like protein) family (Figure 2).
In the case of Fe, transporter activity in delivering the ion to
the root cell is often supplemented by secretion of chelators
which mobilise Fe3+ from the soil. Further insights into the
mechanism of Fe uptake have emerged from the recent
identification of a membrane-bound ferric-chelate reductase
in Arabidopsis ( see Note added in proof). At the root surface,
Fe3+ → Fe2+ reduction reduces affinity for the chelator,
thereby increasing the concentration of Fe2+ for subsequent
uptake. The FRO2 gene was identified on the basis of
homology with human and yeast plasma membrane enzymes
that transfer electrons from FADH2 to an acceptor on the
opposing side of the membrane via two intramembrane haem
groups. FRO2 transcripts accumulate in roots in response to
Fe deficiency. Furthermore, FRO2 complements mutants
which are defective in ferric chelate reductase activity. The
deduced structure of FRO2 contains a cytosolic binding site
for FAD and conserved intramembrane His residues thought
to be involved in coordination of the two haems.
In complementation studies using yeast zrt mutants, four
genes (ZIP1–4), which encode transporters involved in
Zn2+ uptake, were identified in Arabidopsis [14••]. Each of
these transporters is predicted to possess 7–9 transmembrane spans, and expression of two (ZIP1 and ZIP3) is
preferential in root tissue and enhanced by Zn2+ starvation.
These findings suggest that ZIP1 and ZIP3 are involved in
Zn2+ uptake from the soil. The differential substrate specificity and expression patterns of ZIP isoforms might point
to their discrete physiological roles. Competition experiments with other divalent cations also indicate that ZIP1
and ZIP3 are distinctly Zn2–-specific, while ZIP2 exhibits
equal or greater affinity for Cu2+ and Cd2+.
Sequence analysis of ZIP family members demonstrates a
degree of conservation in putative transmembrane spans
IV, V and VI, possibly associated with an intramembrane
metal binding site [15•]. Furthermore, in the majority of
members, a possible heavy metal binding site has been
identified in the extramembrane loop between transmembrane spans III and IV. This conserved sequence has the
form HXHXH, a motif found in another, otherwise unrelated family of heavy metal transporters from bacteria.
A completely different type of transporter involved in
cation uptake has been identified in wheat roots and
shoots. The LCT1 (for low affinity cation transporter) gene
was originally identified by virtue of its ability to restore
growth of a K+ uptake-deficient yeast in low millimolar K+
concentrations [16]. LCT1 was originally shown to transport K+ and Na+ in yeast; however, further work
demonstrated an ability also to transport Ca2+ and Cd2+
[17••]. Although the physiological function of LCT1 is
unknown, this is the first transport system identified as a
candidate for mediating Ca2+ uptake by plant cells.
Our knowledge of the pathways for divalent cation transport
across the plasma membrane therefore remains fragmentary,
yet, significant advances can be expected in the coming
years, not only from complementation screens, but also from
bioinformatic approaches to genome sequences.
Plasma membrane transport in context Maathuis and Sanders
239
Table 2
Gene products involved in the uptake of phosphate in plants.
Protein
Species
Expression
Transport Km
Reference
APT1–2 (APT1–4)
A. thaliana
Root/low level in leaf
–
[33,35•]
PHT1
A. thaliana
Root
3 µM
[34]
Potato
Root/tuber/leaf flower
280 and 130 µM
[31•]
Tomato
Root/mature leaf
31 µM
[30•]
M. trunculata
Root
192 µM
[32•]
StPT1–2
LePT1
MtPT1–2
Potassium transport
To maintain the essential roles that K+ plays in cellular
homeostasis requires sophisticated means to move K+ at
key locations throughout the plant, which include the
soil/root interface, the xylem, and cells involved in movement, for example in guard cells, which are responsible for
the opening and closing of stomatas.
K+ transport at the soil/root interface
The process of K+ uptake in plant roots has been a major
focus of plant physiology. Conventionally, K+ accumulation
at various ambient K+ concentrations is perceived as occurring at the root cell plasma membrane through two or more
independent influx systems with respectively high and low
affinities. Traditionally, low affinity K+ uptake is believed
to be mediated by K+ selective ion channels, a notion supported by both patch clamp characterisation of root K+
channels and yeast complementation studies [2].
Thermodynamic constraints require energisation of high
affinity K+ uptake via carriers [4].
The convenient notion of a channel and carrier mediated
low and high affinity K+ uptake is rapidly crumbling in the
face of recent data. Functional analysis of the role of AKT1
(a K+ selective inward rectifying channel or KIRC) in planta has been advanced [18••] by identifying an AKT1 null
mutant (akt1-1) from an Arabidopsis T-DNA mutagenised
population. Surprisingly, differences in growth between
wild type and akt1-1 were apparent only in the presence of
millimolar ammonium and with K+ levels of >1 mM. In the
presence of ammonium, Rb+ uptake from external solutions was reduced, but progressively so at lower Rb+
concentrations. A reduced Rb+ influx in the mutant also
occurred in the absence of ammonium conditions where no
phenotype was observed. The combined results of this
study indicate that, in the presence of ammonium, the
AKT1 channel plays a role in high affinity K+ uptake.
They also indicate that AKT1 may not be the predominant low affinity K+ influx pathway in A. thaliana roots, as
at 1 mM external Rb+ concentrations the mutant shows
around 70% of the wild-type uptake.
With a sufficiently negative membrane potential, the
influx of K+, even from lower micromolar concentrations,
can proceed ‘down hill’ and this obviates the requirement
for an energised carrier mechanism. Nevertheless, a large
number of carrier type K+ transporters has now been
identified (Table 1, Figure 2). In barley, HvHAK was
cloned [19••] using degenerate primers for conserved
regions of the Schwanniomyces HAK (high affinity K+)
transporter. Complementation of K+ transport deficient
yeast restored high affinity Rb+ uptake, which, in agreement with the observations of Hirsch et al. [18••], was
sensitive to ammonium. The HvHAK1 protein has 12
putative membrane spanning regions and is a member of
a large conserved family of transporters that are highly K+
selective and probably function as H+ coupled systems
[19••]. The barley member of this family shows homology to HAK/KUP transporters found in bacteria
(Escherichia coli), fungi (Schwanniomyces occidentalis),
plants (Lyphopyrum, wheat, rice and Arabidopsis) and animals (Homo sapiens). HvHAK1 transcript is exclusively
found in roots and highly induced by K+ starvation, which
is in agreement with the frequently observed induction of
high affinity K+ uptake in intact plants.
By searching for HAK/KUP homologues in A. thaliana
EST databases, several groups isolated a number of
HAK/KUP isoforms (AtKUP 1–4). The Rb+ and K+ transport capacity of AtKUP1 was analysed in homologous
and heterologous expression systems [20•,21•]. In both
expression systems AtKUP1 mediated high affinity Rb+
transport with a micromolar Km indicating a role in high
affinity K+ transport in vivo. In addition to high affinity
K+ transport, AtKUP1 expressing yeast cells showed a
rise in Rb+ uptake from millimolar concentrations [20•].
The latter suggests that HAK/KUP type proteins may
also contribute to low affinity K+ transport. Expression
patterns of AtKUP1 differ in various reports (Table 1) and
expression of at least one isoform (AtKUP3) is up regulated upon K+-starvation [21•].
A class of putative high affinity K+ transporters, not related to HAK/KUP, is provided by HKT1 type mechanisms
[22]. Originally cloned from wheat, HKT1 catalyses
K+:Na+ (µM ambient Na+) or Na+:Na+ (mM ambient Na+)
symport. Homologues of HKT1 have been found in barley [23], rice [24] and A. thaliana [22] and in both barley
and wheat mRNA levels increase in K+ starvation conditions [23]. The physiological relevance of HKT1 remains
240
Physiology and metabolism
Figure 3
Model according to Doyle et al. 1998 [44] for
the permeation of K+ ions through an inward
rectifying K+ channel with a pore region
similar to that of the Shaker family K+
channels. Ions traverse a narrow pore within
the channel, which forms the selectivity filter
(SF) and can hold two ions simultaneously.
The large aqueous cavity helps to stabilise
cation(s) in the middle of the membrane by
the dipole action of the depicted helices.
SF
–
–
Current Opinion in Plant Biology
to be clarified but may be in providing a pathway for Na+
entry into the plant [24].
K+ release into the xylem
SKOR [25••] is an Arabidopsis K+ selective channel and a
member of the Shaker gene family (Figure 3). Shaker type
channels are K+ selective, voltage gated outward rectifying
channels that were first identified in Drosophila shaker
mutants. In spite of the large degree of homology to KAT
and AKT inward rectifying channels, SKOR mediates K+
efflux. How such similar primary protein structures result
in K+ channels with contrasting voltage sensitivity remains
to be explained. Expression patterns and gene disruption
mutants imply a clearly defined function for SKOR in
xylem loading of K+: GUS constructs with the SKOR promoter show expression restricted to the root pericycle and
xylem parenchyma cells. Disruption of the SKOR gene did
not affect root K+ levels but caused a decrease in xylem sap
K+ and a reduction in shoot K+ contents, consistent with a
function in K+ release into the xylem. Exposure of plants
to the stress hormone ABA, which is believed to play a role
in ion translocation to the shoot, resulted in a rapid
decrease of SKOR transcript.
K+ transport in guard cells
The first plant K+ channels were identified in guard cells
which control stomatal aperture by osmotic swelling and
shrinking caused by the movement of large amounts of
K+. Via the membrane voltage, either inward or outward
rectifying channels are activated to allow a sustained inward
or outward flux of K+ for stomatal opening or closing,
respectively. A variety of stimuli such as ABA, CO2 and
oxidative stress promote stomatal closure, probably via a
raise in cytosolic Ca2+. A direct target for cytosolic Ca2+
Figure 4
NH2
COOH
Current Opinion in Plant Biology
Generalised model for the topology of high
affinity phosphate carriers. Transmembrane
spans show an internal repeat and are
arranged in a typical 6+6 structure
characteristic of many carriers belonging to
the major facilitator superfamily. The large
cytoplasmic loop contains a putative
phosphorylation site for kinase C.
Plasma membrane transport in context Maathuis and Sanders
has now been described in Vicia faba guard cells [26] as a
Ca2+ dependent protein kinase that phosphorylates and
down regulates KAT1, the predominant KIRC in guard
cell plasma membranes.
241
result from the use of Saccharomyces as expression system
where the interaction of several proteins is necessary for the
proper functioning of high affinity phosphate transport [36].
Cytosolic Ca2+ also affects the activity of a newly identified
K+ selective outward rectifying channel (KORC) [27]. In
contrast to previously described KORCs that show sustained currents, this particular type of KORC rapidly
inactivates. The physiological role of this channel remains
to be elucidated, but may be in the transduction of signals
during stomatal closing. Additional potential modulators of
stomatal aperture that act on K+ channels are agents that
affect the polymerisation of actin filaments [28] and the
osmolarity of solutions facing the membrane [29].
The relative contribution of these gene products to the
overall uptake of phosphate by plants will be affected by
the formation of mycorrhizae, which can drastically
improve plant phosphate nutrition. The presence of mycorrhizae will shift phosphate uptake to GvPT-type
transporters at the soil/fungus interface and can reduce the
amount taken up by the plant root to almost nil. The
mechanism of phosphate transfer from fungus to root
remains unknown but probably involves separate gene
products as was shown for Medicago where MtPT transcript
was significantly reduced after mycorrhiza formation [32•].
Phosphate uptake in plant roots
Nitrate uptake
Phosphate is an essential macronutrient and plant cells control cytosolic phosphate within narrow limits by balancing
import and export. Rates of phosphate uptake depend on
growth demand and phosphate supply, the latter frequently being inadequate due to the low mobility of phosphate
in the soil. At the prevailing soil pH, phosphate is most
readily taken up by plants as H2PO4–. External concentrations of this nutrient are typically a few micromolar in
physiological conditions, whereas cytosolic levels are a
1000-fold higher. Phosphate transport shows a pronounced
pH optimum, is highly sensitive to uncouplers [30•] and is
generally assumed to occur via H+ coupled symport.
Nitrate uptake from the soil has long been suspected,
from kinetic studies, to involve an array of different transport systems. Thus, constitutive high affinity and
non-saturable kinetic phases exist in roots along with a
high affinity phase, which is induced by nitrate [38]. The
properties of the transport systems that underlie this
kinetic complexity are beginning to be elucidated with
candidate genes falling into two families.
Several genes encoding high affinity phosphate transporters have been cloned (Table 2; [30•–32•,33,34,35•]).
The proteins encoded by these genes are around 60 kD,
and show high levels of homology to each other and to the
high affinity phosphate transporters of yeast PHO84 [36]
and mycorrhizal fungi GvPT [37]. APT1 and APT2 [35•] for
example, are both expressed in Arabidopsis root tissue and
share 99% identity in their coding regions; however, their
promoter regions are very different, pointing to cell-typespecific or development-specific expression for either gene.
Hydrophobicity plots show a common structure of
12 transmembrane spans that contain an internal repeat of
6 + 6 transmembrane spans (Figure 4) with a large central
hydrophobic region protruding into the cytosol that carries
a highly conserved putative kinase phosphorylation site. In
some cases transcript levels were shown to be sensitive to
the phosphate status of the plant (e.g. for APT1, APT2)
whereas other genes appear to be constitutively expressed
(e.g. for StPT2).
The functional analysis of LePT1 from tomato [30•] and of
MtPT1 from Medicago [32•] was carried out in the yeast
pho84 strain. In both cases, yeast growth was restored on
micromolar phosphate and transport assays confirmed the
restoration of phosphate uptake with an apparent Km of 31
µM for LePT1 and 192 µM for MtPT1. Both Km values are
much higher than those established in intact plants and may
The first family of nitrate transporters is identified by a
consensus motif (FYXXINXGSL), characteristic of the
PTR family of peptide transporters present in yeast,
plants and mammals. At least two members of this family are present in Arabidopsis: NRT1 (or CHL1) and
NRT3 (or NLT3) [38]. NRT1 expression is inducible by
nitrate, whereas that of NRT3 appears to be constitutive.
Until recently both transport systems were thought to be
involved only in low affinity transport. Careful analysis
of one chl1 mutant line containing an active transposable
element in CHL1, however, revealed defects in both low
and high affinity transport [39••]. The extent to which
CHL1 participates in high affinity transport depends
critically on growth conditions: the presence of ammonium ions results in a marked contribution of CHL1 to
high affinity uptake, whereas in the absence of ammonium no contribution is apparent. CHL1, therefore,
appears to behave as a dual affinity transporter, a phenomenon that can be explained by random binding of H+
and nitrate to the carrier [40].
A CHL1 homologue from Brassica napus has been functionally characterised in oocytes with respect to substrate
specificity [41••]. Generally, transport carriers exhibit a
high degree of substrate specificity and it was, therefore,
surprising to find that CHL1 not only translocates nitrate,
but also histidine and to a lesser extent, other basic amino
acids. The physiological significance of this observation
is not known but could relate to delivery of amino acids
to growing cells. Resolution of expression patterns at a
cellular level could help resolve this issue; it is already
known that two putative nitrate transporters in the PTR
242
Physiology and metabolism
family exhibit differential expression patterns in tomato
roots [42].
Members of the second nitrate transporter family (NRT2)
are nitrate inducible, belong to the major facilitator superfamily and were identified originally on the basis of
homologies to fungal and algal nitrate transporters [38].
Confirmation of a role in nitrate transport awaits their functional expression or the identification of mutants in these
genes. The high degree of correlation between expression
levels of the tobacco transporter NRT2 with high affinity
nitrate transport activity, however, provides evidence for a
function in inducible, high affinity nitrate transport [43•].
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
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Marschner H: Mineral Nutrition of Higher Plants. London: Academic
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Korthout HAAJ, deBoer AH: A fusicoccin binding-protein belongs
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Conclusions and prospects
The number of transporters identified at a molecular
level has increased dramatically over the past years. For
any given nutrient, an array of potential transport pathways is present at the plasma membrane, even within a
single species.
At one level, this complexity reflects the presence of isoforms that are often expressed on a tissue- or cell
type-specific basis. Isoforms enable transport to be controlled at a transcriptional level in response to
developmental or environmental signals. At a second
level, different classes of transporter are apparent for ions
like K+ and nitrate which may reflect the wide range of
nutrient concentrations to which plants are exposed.
Although the complexity of multiple transport pathways
will doubtless increase as genomic information expands,
we can expect marked advances in our understanding of
the functional attributes of transporters with the combination of reverse genetic and physiological approaches over
the coming years.
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In this study, detergent-solubilised plasma membrane H+-ATPase isolated
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H+-ATPase was recovered in a nonactivated form.
Considerable effort and expense is required for the inorganic fertilisation of many agricultural systems. Many
such systems are, indeed, over-fertilised, with devastating
consequences for water courses which become eutrophic.
Identification of those transporters which are pivotal in
the uptake of inorganic nutrients from the soil and the
subsequent redistribution of nutrients around the plant
offers the exciting possibility of engineering more efficient strategies for fertiliser application. Such strategies
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12. Baunsgaard L, Fuglsang AT, Jahn T, Korthout HAAJ, deBoer AH,
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By testing the fusicoccin binding activity of yeast membranes, the carboxy-terminal regulatory domain of AHA2 was found to be part of a functional fusicoccin
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9.
•
10. Oecking C, Piotrowski M, Hagemeier J, Hagemann K: Topology and
•
target interaction of the fusicoccin-binding 14-3-3 homologs of
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Experiments carried out with sealed plasma membrane vesicles show that
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•• of a family of zinc transporter genes from Arabidopsis that respond
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Report on the identification of ZIP genes from Arabidopsis as encoding Zn2+
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The plant ZIP transporters are related to metal transporters found in many other
eukaryotes, including yeast, protozoa and — more distantly — humans.
Note added in proof
15. Eng BH, Guerinot ML, Eide D, Saier MH: Sequence analyses and
•
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17.
••
18. Hirsch RE, Lewis BD, Spalding EP, Sussman MR: A role for the AKT1
•• potassium channel in plant nutrition. Science 1998, 280:918-921.
Reverse genetic screening identified an Arabidopsis thaliana mutant in
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inward-rectifying potassium channels and displayed reduced potassium
(rubidium-86) uptake in the presence of ammonium.
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•• HAK1 gene of barley is a member of a large gene family and
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K+ transporter that is expressed in root cells and which shows a high degree
of homology to bacterial high affinity K+ transporters.
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Arabidopsis. Plant Cell 1998, 10:63-67.
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•
gene encoding high-affinity potassium transport activity. Plant
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of non-plant Kup and of HAK1 potassium transporters from Escherichia coli
and Schwanniomyces occidentalis. AtKUP1 and AtKUP2 are able to complement a potassium transport deficient E. coli triple mutant. Kinetic characterisation and expression patterns are reported. Through sequence analysis a family
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24. Golldack D, Kamasani UR, Quigley F, Bennett J, Bohnert HJ: Salt
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25. Gaymard F, Pilot G, Lacombe B, Bouchez D, Bruneau D, Boucherez J,
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release into the xylem sap. Cell 1998, 94:647-655.
SKOR, a K+ channel identified in Arabidopsis, displays the typical hydrophobic
core of the Shaker channel superfamily. Expression in Xenopus oocytes identified SKOR as the first member of the Shaker family in plants to be outwardly
rectifying. SKOR expression is localised in root stelar tissues and evidence indicates that SKOR is involved in K+ release into the xylem sap toward the shoots.
26. Li J, Lee YR, Assmann SM: Guard cells possess a calcium-dependent
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•
analysis and cell-specific expression of a phosphate transporter
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of 31 µM and is highly dependent on the external pH. The work presents
molecular and biochemical evidence for distinct root cells playing an important role in phosphate acquisition at the root/soil interface.
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