Download Partitioning of nutrient transport processes in roots

Journal of Experimental Botany, Vol. 52, Roots Special Issue,
pp. 445±457, March 2001
Partitioning of nutrient transport processes in roots
Mark Tester1 and Roger A. Leigh
Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK
Received 23 June 2000; Accepted 1 September 2000
Abstract
Roots have a range of cell types that each contribute
to the acquisition of nutrients and their subsequent
transfer to the xylem. The activities of these cells
must be coordinated to ensure that delivery of nutrients to the shoot occurs at a rate that matches
the demands of growth. The partitioning of transport
processes between different cell types is thus
essential for roots to function effectively. This partitioning is considered at the level of proteins,
organelles and cells in relation to the accepted
concepts of how nutrients are taken up by roots
and delivered to the xylem. Using K+ as an example,
the evidence underpinning current concepts is
examined, gaps in understanding identified and the
contribution of some new approaches assessed.
Key words: Root physiology, nutrient uptake, stress
tolerance, cell-specific expression, transgenic plants.
Introduction
The ability of roots to obtain nutrients from the soil
and to deliver these to the aerial tissues at a rate that
matches the needs of growth is key to ensuring that
the shoot has the resources it needs to function effectively. When roots fail to do this, plants show a range of
visual symptoms indicative of nutrient de®ciencies (e.g.
smaller stature, discolouration). However, even before
such obvious symptoms appear, mismatches between the
demands of the shoot and the supply from the roots can
affect productivity. In agricultural systems this results in
smaller yields, while in natural ecosystems it may affect
the ability of plants to compete effectively.
In general, it is understood how roots take up and
transfer nutrients to the xylem for onward transport to
1
the shoot. They form highly branched structures that
provide a large surface are for nutrient and water
absorption and which ramify through the soil pro®le to
tap new sources of nutrients. Further, this structure is
dynamic; it can respond to local conditions, for example,
becoming highly branched in regions of high nutrient
availability (Drew and Saker, 1975), at least for some
nutrients in some species (Robinson, 1994). The activity
of roots also affects, in a number of ways, the availability
of nutrients at the soil surface. Thus the rate at which
nutrients and water are taken up is important in generating the driving forces for movement of nutrients
through the soil either by mass ¯ow of soil solution or
by diffusion (Tinker and Nye, 2000) while secreted
chemicals can modify the root environment to increase
nutrient solubility or promote the formation of bene®cial
associations with micro-organisms such as rhizobia or
mycorrhizal fungi.
Once ions reach the root surface, they are transferred
across the root to the xylem by apoplastic (extracellular)
anduor symplastic (intracellular) pathways (Clarkson,
1993). The apoplastic route can be followed as far as
the endodermis where the presence of a poorly ionpermeable secondary thickening in the cell wall (the
Casparian Strip) restricts further movement. Thus any
nutrient which reaches this point via the apoplastic
pathway must, perforce, cross the plasma membrane of
the endodermal cells to reach the stele. However, entry
into the symplastic pathway is not restricted to the endodermis. It can also occur in root hairs, other epidermal
cells, and in the cortex. Once in the stele, ions are secreted
into the xylem for transfer to the shoot.
Although this general picture is widely accepted, the
molecular processes that mediate and control uptake and
trans-root movement of nutrients are not well understood, nor are the activities of individual cells in these
processes completely described. A key to understanding
root function is to describe how processes that contribute
To whom correspondence should be addressed. Fax: q44 1223 333953. E-mail: [email protected]
ß Society for Experimental Biology 2001
446
Tester and Leigh
to the uptake of nutrients and their transfer to the xylem
are partitioned at different levels. This partitioning is
considered in an hierarchical way here by examining it at
the level of proteins within membranes, compartments
within cells, and cell types within roots. For each level of
organization present concepts and understanding are
outlined, what evidence supports them is considered,
what gaps in knowledge exist, and how new approaches
could help to provide new insights and understanding.
Particular emphasis has been placed on Kq, but the
general concepts are applicable to other ions.
Partitioning of transport between proteins
within membranes: K+ uptake as a paradigm
Roots are able to absorb nutrients over a wide range of
external ion concentrations and, provided the rate of delivery to the root surface is high enough, plants can grow
at maximal rates in the presence of very low external
nutrient concentrations. This is exempli®ed by the experiments of Asher and Ozanne who grew a number of
plants in rapidly ¯owing nutrient solutions (delivered at
1000 l pot 1 d 1) containing a range of Kq concentrations (Asher and Ozanne, 1967). Their results showed that
individuals of all of the species they used could survive in
1 mM Kq, a few grew maximally in 8 mM Kq, while the
majority of the remainder needed only 24 mM Kq for
optimal growth. Further, very large changes in the
external concentration were not matched by proportionately large changes in shoot or root Kq concentrations
(Table 1). A 1000-fold increase in external Kq concentration (from 1 to 1000 mM) resulted in only a 6- to 14-fold
change in the total Kq concentration in the roots and
shoots. Additionally, the root and shoot Kq concentrations were comparable in all of the species, implying
that Kq uptake and transfer to the shoot are regulated in
a broadly similar way, at least in terms of the outcome
(i.e. the ®nal root and shoot Kq concentrations) that is
achieved. These observations indicate that Kq uptake by
roots can operate at very low concentrations and that the
rate of uptake is matched to growth of the shoot and the
root to regulate tissue Kq concentrations.
Net Kq uptake at the root plasma membrane is
classically viewed as resulting from the operation of active
and passive transporters, which have different af®nities
for Kq and which make different contributions to uptake
as the external concentration changes. Evidence for the
operation of different Kq uptake systems came from
the work of Epstein and co-workers (Epstein et al., 1963;
Welch and Epstein, 1968) who suggested that at low
external Kq concentrations (below approximately 1 mM),
uptake of Kq is mediated by a high-af®nity saturable
mechanism (so-called System 1) that is highly selective
for Kq. At higher external Kq concentrations, a second,
less selective system operates (System 2).
De®ning the precise nature of the transport systems
that are responsible for the observed patterns of uptake
requires knowledge of the way electrochemical potential gradients for Kq respond to the changes in external
Kq. Detailed studies in Arabidopsis roots (reviewed by
Maathuis and Sanders, 1996) showed that growth and Kq
uptake were possible at an external Kq concentration
of 10 mM and that, at the prevailing membrane potential difference (PD) of 129 to 153 mV, and cytosolic
Kq activity (80 mM), Kq uptake into root epidermal
cells at this external Kq concentration has to be active
(Maathuis and Sanders, 1993). Patch clamp studies
showed that this uptake is probably mediated by
a Kq: Hq symporter (Maathuis and Sanders, 1994).
Uptake within the concentration range of System 2 is
probably passive and mediated by inward Kq-selective
channels. Patch clamp studies have indicated that such
channels are present in epidermal and cortical cells from
Table 1. Comparison of shoot and root Kq concentrations in plants growing in solutions containing 1 or 1000 mM Kq
Values in brackets are the fold increase of Kq concentration in the shoots and roots of plants grown in 1000 mM Kq compared with those grown
in 1 mM Kq. Data are from Asher and Ozanne (Asher and Ozanne, 1967), with shoot Kq concentration recalculated on the basis of tissue water from
the results in their Tables 3 and 4.
Species
Trifolium subterraneum
Trifolium hirtum
Ornithopus sativus
Vicia sativa
Erodium botrys
Lolium rigidum
Vulpia myuros
Hordeum vulgare
Bromus rigidus
Avena sativa
Shoot Kq concentration (mM)
Root Kq concentration (mM)
wKxo ˆ 1 mM
wKxo ˆ 1000 mM
wKxo ˆ 1 mM
wKxo ˆ 1000 mM
23
29
25
26
23
23
38
15
35
30
267
229
213
160
177
238
240
215
274
204
20
19
18
13
11
15
17
10
14
13
150
130
106
92
89
131
110
135
142
114
(11.6)
(7.9)
(8.5)
(6.2)
(7.7)
(10.3)
(6.3)
(14.3)
(7.8)
(6.8)
(7.5)
(6.8)
(5.9)
(7.1)
(8.1)
(8.7)
(6.5)
(13.5)
(10.1)
(8.8)
Partitioning of transport in roots
a range of species (Schachtman et al., 1991; Maathuis
and Sanders, 1995; Roberts and Tester, 1995; Gassmann
and Schroeder, 1994). In Arabidopsis root cells there are
at least two such channels with conductances of 5 pS and
20 pS, respectively, when measured with 10 mM Kq on
each side of the membrane. The properties of the 5 pS
channel are consistent with it being the dominant pathway for Kq uptake at mM external concentrations of
Kq and its activity increases following Kq starvation
(Maathuis and Sanders, 1995). Similar results have been
observed in wheat roots where there was a 6- to 10-fold
increase in a channel-mediated Kq transport following
growth in Kq-free media (Buschmann et al., 2000),
suggesting an important role for Kq channels in Kq
nutrition. Other Kq-permeable channels have also been
described in roots, for example, non-selective cation
channels (Amtmann and Sanders, 1999; Davenport and
Tester, 2000) and cyclic-nucleotide-gated channels
(KoÈhler et al., 1999; Leng et al., 1999), but their
signi®cance in overall Kq uptake into the root is
unknown.
The identi®cation of the proteins responsible for
active and passive uptake of Kq has progressed rapidly
in the last decade mainly through the use of heterologous
expression systems both to clone genes and to characterize their products. A number of plant Kq transporters
have been identi®ed by functional complementation of
the Kq transport-de®cient trk1 trk2 mutant of yeast
(Saccharomyces cerevisiae). This mutant is unable to grow
at low external Kq concentrations and plant cDNAs
encoding Kq transporters have been identi®ed by their
ability to restore growth of the mutant on low Kq. Further characterization has been performed using a variety
of heterologous expression systems such as yeast itself,
Xenopus oocytes, and insect cells. Transporters that
have been identi®ed include inwardly- and outwardlyrectifying Kq-selective channels, and high af®nity active
transporters (Rodriguez-Navarro, 2000; Schachtman,
2000). Among those that have been shown to be
expressed in roots are AKT1 (an inward rectifying
channel; Sentenac et al., 1992; Lagarde et al., 1996),
SKOR (an outward rectifying channel; Gaymard et al.,
1998), HKT1 (initially identi®ed as a Hq-linked Kq
symport, but now thought to function as a Naq: Kq or
Naq: Naq symport; Schachtman and Schroeder, 1994;
Rubio et al., 1995; Gassmann et al., 1996), and members
of the KUPuHAK family (dual af®nity transporters
with similarity to fungal Hq: Kq symporters; Kim
et al., 1998; Fu and Luan, 1998; Santa-Maria et al.,
1997; Rubio et al., 2000).
AKT1 is a member of the Shaker superfamily of
voltage-gated Kq-selective ion channels that in animal
cells mediate outward Kq transport (Jan and Jan, 1997).
AKT1 shares many of the structural features of the
animal channels, including six membrane spanning
447
domains (S1±S6), a preponderance of positive charges in
S4 that probably represents the voltage-sensing domain,
and a hydrophilic domain (P-domain) between S4 and S5
that forms part of the ion-conducting pore and selectivity
®lter and contains a conserved GYGD motif (Sentenac
et al., 1992). Despite this high level of structural similarity, electrophysiological studies of AKT1 expressed in
insect cells indicate that it functions as an inward rather
than an outward rectifying Kq channel (Gaymard et al.,
1996). Localization using the GUS reporter gene indicate
that AKT1 is expressed mainly in root epidermal and
cortical cells and thus could catalyse passive Kq uptake
(Lagarde et al., 1996). This was con®rmed using an
Arabidopsis AKT1 knock-out mutant (akt1-1). Patch
clamp analysis of root cells from this mutant showed
that they lack inward Kq currents and are compromised
in Kq uptake (Hirsch et al., 1998; Spalding et al., 1999).
SKOR is structurally similar to AKT1, but has been
shown to mediate outward rather than inward Kq
transport and to be expressed speci®cally in stelar cells.
It is thus thought to be involved in the passive transport
of Kq out of the stelar cells into the xylem. Knockout
mutants lacking SKOR have diminished Kq transport to
the shoot and this defect can be complemented by
reintroduction of the SKOR gene (Gaymard et al., 1998).
HKT1 was cloned from wheat and, in roots, is
preferentially expressed in cortical cells. Its gene product
was initially characterized as a Hq : Kq symport that can
mediate high af®nity active transport of Kq when
expressed in Xenopus oocytes or yeast (Schachtman and
Schroeder, 1994). However, subsequent work showed
that it probably functions as a Naq: Kq symport at
low external Naq concentrations and as a Naq: Naq
symport at higher external Naq concentrations
(Gassmann et al., 1996; Rubio et al., 1995). Thus its
precise role in Kq transport remains somewhat equivocal. It has been shown that Naq-dependent Kq uptake
does not occur in roots of terrestrial plants (Maathuis
et al., 1996) and that an Arabidopsis HKT1 orthologue
encodes a Naq transporter that is unaffected by Kq
(Uozumi et al., 2000). Thus the prevailing evidence tends
to support the view that HKT1 may be more important in
Naq uptake. Nonetheless, its involvement in high af®nity
Kq transport is supported by the observation that its
expression is rapidly up-regulated in response to removal
of external Kq and with a time-course that matches
changes in Kq uptake (Wang et al., 1998). In addition,
Spalding et al., using the Arabidopsis akt1-1 knockout
mutant, found that high af®nity non-AKT1-dependent
Kq uptake in the roots of these plants was sensitive to
the external concentration of both Hq and Naq which
might indicate a role for HKT1 in this process (Spalding
et al., 1999). Sensitivity of HKT1 to NH4q is consistent
with this suggestion, but it has not been de®nitely proven
(Santa-Maria et al., 2000).
448
Tester and Leigh
Potassium transporters of the KUPuHAK subtypes
have been isolated from a variety of plants and their
structural and sequence similarities indicate that they
form a family of related transporters (Rubio et al., 2000).
They have 12 membrane-spanning domains and a long
cytosolic C-terminal domain (Kim et al., 1998; Fu and
Luan, 1998; Santa-Maria et al., 1997; Rubio et al., 2000).
Up to 13 related genes are present in Arabidopsis and
members of the family have been detected in a number of
other plants (Rubio et al., 2000). The proteins are predicted to be about 30% identical in amino acid sequence
to both the KUP Kq transport system from E. coli and
the HAK1 system from Schwanniomyces occidentalis.
The fungal HAK1 gene products encode high af®nity
Hq: Kq symporters (Haro et al., 1999) and the plant
orthologues mediate high-af®nity Kq transport when
expressed in yeast (Kim et al., 1998; Fu and Luan, 1998;
Santa-Maria et al., 1997; Rubio et al., 2000). However,
it is unclear whether the transport catalysed by the
plant members of this family is active or passive as
attempts to characterize the gene products by expression
in Xenopus oocytes (which are more amenable to
electrophysiology than yeast) have failed (Kim et al.,
1998; Rubio et al., 2000). Evidence that this transport
may be channel-like is suggested by the ability of KUP1
to catalyse high rates of Kq transport at mM concentrations, the sensitivity of the transport to tetraethylammonium (a blocker of Kq channels) and by the
presence of an IYGD motif with homology to the GYGD
that is characteristic of Kq channels (Fu and Luan, 1998).
KUPuHAK transporters may also be involved in the
NH4q-inhibited, high af®nity Kq transport because the
activity of these transporters is sensitive to NH4q (SantaMaria et al., 1997, 2000), although they lack the Naq
sensitivity that the NH4q-inhibited component was
reported to have (Spalding et al., 1999).
How Kq uptake is partitioned between different transporters remains unclear. The isolation of genes for both
Kq channels and putative high af®nity active transporters
appears to con®rm that the elements needed to explain
Systems 1 and 2 are present. However, Sanders showed,
using reaction kinetic modelling, that a single transporter
can give rise to multiphasic uptake kinetics and so
multiple transporters are not a prerequisite for such
kinetics (Sanders, 1990). Similarly, energy-barrier models
can also explain predict multiphasic kinetics (White and
Ridout, 1995). A simple explanation that assigns transport by HKT1, HAK and KUP transporters to System 1
and that by AKT1 or other ion channels to System 2 is
complicated by observations that suggest that all of these
transporters may be capable of mediating transport at
both mM and mM external Kq concentrations. For
instance, using an AKT1 knockout mutant (akt1-1),
evidence was found to suggest that this channel accounts
for a signi®cant proportion of total Kq uptake by
Arabidopsis roots at external Kq concentrations as
low as 10 mM (Hirsch et al., 1998; Spalding et al.,
1999). This need not indicate that AKT1 mediates an
active transport process since passive in¯ux can occur at
such low external concentrations if the PD is suf®ciently
negative. With a cytosolic Kq activity of about 80 mM
(Maathuis and Sanders, 1993; Walker et al., 1996),
passive inward transport of Kq could be achieved from
solutions containing 8 mM Kq if the PD was about
236 mV or more negative. Plant cells do not normally
have such negative PDs (Maathuis and Sanders, 1993),
but values have been reported of around 225 mV in
Arabidopsis roots and so, under these conditions, AKT1
could mediate passive inward Kq transport from mM
external Kq (Hirsch et al., 1998). Similarly, while
transporters of the KUPuHAK family are capable of
high af®nity Kq transport (Santa-Maria et al., 1997;
Kim et al., 1998; Fu and Luan, 1998; Rubio et al., 2000),
KUP1 has been reported to mediate high rates of Kq
transport from solutions containing up to 150 mM Kq
when expressed in yeast (Fu and Luan, 1998). However,
the reliability of the yeast trk1 trk2 mutant for making
Kq uptake measurements at high external Kq concentrations has been questioned because the mutant has a high
intrinsic capacity for transport at these elevated concentrations (Rubio et al., 2000). These authors suggest that,
under these conditions, assigning differences in Kq
uptake to a heterologously expressed protein may be dif®cult. Therefore, the role of the HAKuKUP transporters
in passive Kq uptake remains to be established.
The importance of the PD in controlling directions of
ion movement must also be emphasized. The activity of
ion channels in catalysing inward and outward movements of Kq, and therefore the direction of rapid passive
movements of Kq, are controlled by PD, a parameter
which is, in turn, the output of a complex interaction of
®xed charges and membrane transport processes (Nobel,
1999). The role of PD in Kq transport has been examined
for guard cells (Thiel et al., 1992), but there has been very
little such work in roots (Wegner and de Boer, 1997).
PD was measured across the plasma membrane of the
cortical and stelar cells of maize roots and it was found
that ABA caused the stelar cell PD to become more
negative relative to the xylem apoplast (Roberts and
Snowman, 2000). Thus, the driving force favouring movement of Kq out of the stelar cells is less with ABA
(assuming there were no effects on the transmembrane
Kq concentration gradient). These results are consistent
with a role for PD in inhibiting the loading of solutes into
the stelar apoplast, for subsequent transfer to the xylem
and then shoot. This is in addition to the inhibition by
ABA of the conductance of the plasma membrane to Kq
(Roberts, 1998) and Cl (Gilliham and Tester, 2000).
There are undoubtedly more Kq transporters to be
discovered in plants and some of these will mediate
Partitioning of transport in roots
q
K uptake processes in roots. The sequencing of the
Arabidopsis genome raises the possibility of identifying
all Kq transporters, at least where they share sequence
homologies with those from other organisms. However,
to characterize their physiological roles it will be necessary to functionally characterize each gene product in
heterologous systems to gain knowledge of their properties, and then to con®rm their role in Kq acquisition by identifying which transporters are expressed in
roots, which cells and membranes they are located in,
what conditions they are expressed in, and what the
consequences are of knocking out their expression using
antisense, insertional mutagenesis, gene silencing or
homologous recombination.
449
in the vacuole may re¯ect available supply rather than
export across the tonoplast from a previously replete
store. Therefore, experiments are needed to determine
what happens to vacuolar Kq when replete cells are
placed in de®cient conditions. For nitrate this does lead
to a loss of nitrate from the vacuole (van der Leij et al.,
1998), but this has not yet been tested for Kq. It is
possible that limitations on Kq mobilization from the
vacuole may be imposed by the availability of other
cations to replace it because of the need to maintain
vacuolar sap osmotic pressure (Leigh and Wyn Jones,
1984). If other cations are not available, Kq mobilization
may be limited unless other solutes such as sugars can be
transferred to the vacuole. However, when Kq is replaced
by uncharged solutes, there must also be mobilization of
Partitioning of nutrient ions between the
cytosol and vacuole
Compartmentation of ions in the vacuole has received
much attention because of the importance of this organelle both as a permanent repository for toxic ions or as
a temporary storage site for those nutrients taken up
in excess of immediate needs (e.g. nitrate and phosphate).
In relation to uptake and transfer of nutrients to the
xylem, vacuoles are a side-branch off the main transport
pathway. Nonetheless, the tonoplast has a potentially
important role in regulating ion concentrations in the
cytosol and hence in the symplastic pathway.
Measurements with ion-selective microelectrodes indicate that the concentrations of both Kq (Walker et al.,
1996) and NO3 (Miller and Smith, 1996) are regulated
in the cytosol of root cells while concentrations in the
vacuole vary with external supply, demonstrating the
¯exibility of the vacuole as a storage organelle (Walker
et al., 1996; Zhen et al., 1991; van der Leij et al., 1998). In
the case of Kq, vacuolar Kq activity (aK) in root cells is
about 100 mM in Kq-replete cells, but falls to 10 mM or
less in starved cells (Fig. 1; Walker et al., 1996). In contrast, the aK in the cytosol is relatively constant at about
80 mM over a wide range of Kq supplies, indicating the
tight regulation that is exerted on this parameter (Leigh
and Wyn Jones, 1984). However, the degree of control
differs in different cell types so that at low tissue Kq
concentrations, there is a larger decline in cytosolic aK
in epidermal cells than in cortical cells (Fig. 1; Walker
et al., 1996).
How cytosolic aK is regulated remains unclear. It is
uncertain whether it primarily involves plasma membrane
or vacuolar transport systems or both. The maintenance
of a relatively constant cytosolic aK while that in the
vacuole declines might indicate that Kq is transferred out
of the vacuole to maintain the level in the cytosol.
However, most experiments are done by growing roots
at different external Kq concentrations where the level
Fig. 1. The effect of Kq starvation on Kq activity (aK) in the vacuole
(open symbols) and cytosol (closed symbols) of (A) epidermal and
(B) cortical cells of barley roots. Note that, in both cell types, aK in
the vacuole decrease more than that in the cytosol, and that at low
tissue Kq concentrations there is a larger decline in cytosolic aK in the
epidermal cells than in the cortex. Measurements were made with
Kq-selective microelectrodes (from Walker et al., 1996, where further
details can be obtained).
450
Tester and Leigh
a counteranion along with Kq to maintain electrical
neutrality.
One aspect of the accumulation of Kq in vacuoles
that has received little attention is the observation that
the maximum concentration to which it accumulates
is different in roots and leaves. In many plants under
K-replete conditions, the Kq concentration in the shoots
is approximately twice that in the roots (data in Table 1
for plants grown in 1000 mM Kq). Measurements with
Kq-selective microelectrodes indicate that cytosolic aK
is similar in cells of roots (Walker et al., 1996) and leaves
(T Cuin, SA Laurie, AJ Miller, RA Leigh, unpublished
results) so the differences in the vacuole must result from
differences in the regulation of Kq transport at the tonoplast. Unfortunately, investigations into the nature of
this regulation must await identi®cation of the mechanisms responsible for Kq transport into the vacuole,
which remain elusive (compare Davies et al., 1992; Ros
et al., 1996).
As the symplastic pathway of trans-root transport to
the xylem involves the linked cytosols of adjacent cells,
the concentrations of ions in this compartment presumably exert some in¯uence on the rate of transfer. However, although Kq and N are needed in the shoot in
roughly equal proportions, the concentrations of nitrate
and Kq in the cytosol are very different. Thus, whereas aK
is about 80 mM (Fig. 1), nitrate activity is only about
4 mM (Zhen et al., 1991; Miller and Smith, 1996). Thus,
assuming the two ions move at equal rates through the
symplast (a process that will be primarily determined by
cytoplasmic streaming and transpirationally-driven bulk
¯ow, not diffusional processes) and that the Kq ef¯ux
mechanism controlling release to the xylem responds
linearly to increasing Kq (which is possible, given the
relatively low af®nity of some plant cation channels, e.g.
White and Ridout, 1995), approximately 20-fold more
Kq than nitrate would reach the xylem, at least in plants
performing most of their nitrate reduction in the shoot.
Clearly this level of imbalance does not occur so
mechanisms must exist to modify the relative rates of
loading of these ions into the xylem from the symplast.
Partitioning between cell types within roots
Root structure is complex, with a wide diversity of cell
types apparent even with low resolution microscopy. This
complexity is, of course, a necessary consequence of the
growth and physiology of the root. Many cell types have
particular roles in the uptake of ions and their transfer to
the xylem and these will be discussed with particular reference to root hairs, and cells of the cortex and
the stele.
Trichoblasts, root epidermal cells with an elongated
projection (`root hair') that extends into the soil, are the
most obvious root cell type in which function is intuitively
distinct. Root hairs appear to be specialized for the
uptake of nutrients from the soil because they increase the
surface area for absorption. The result of their activity
is effectively to move the zone of depletion of nutrients
further from the root surface (Tinker and Nye, 2000).
This has the effect of increasing the volume of soil
exploited for a given root length, whilst minimizing the
input of resources needed to achieve this. However, the
zones of depletion of individual root hairs overlap
substantially indicating that more are produced than is
theoretically needed to maximize nutrient availability.
This may be because root hairs are short-lived and must
maximize depletion before they die, but it could also
indicate that they have other functions such as anchoring
the root to enable the root tip to push forward through
the soil without causing the backwards movement of
mature root (Stolzy and Barley, 1968), maintaining
physical contact between roots and soil (Russell, 1977),
and in root exudation (Bhat et al., 1976). The relative
importance of these functions has not been established.
Now that mutants are available that are either compromised in root hair formation or that produce an overabundance of root hairs (Schiefelbein and Somerville,
1990; Grierson et al., 1997), it should be possible to take a
more analytical approach to the role of root hairs and
test theoretical predictions that their major role is in the
uptake of poorly mobile nutrients.
When there is a depletion zone around the root, the
cortex is unlikely to play a signi®cant role in the initial
uptake of nutrients, but when nutrients are at high
concentrations (e.g. after fertilizer application, in a high
nutrient patch, or with toxic concentrations of salts),
solutes could also be taken up by cortical cells. However,
the signi®cance of the exodermis in limiting apoplastic
entry into the cortex is still uncertain, although it can
clearly limit such ¯ow in some species (Peterson, 1987;
Varney et al., 1993). Generalizing from such studies is,
however, not possible, as there is clearly much variation
in exodermal development (Canny and Huang, 1994). For
example, salinity has been reported to induce the formation of an exodermis in cotton (Reinhardt and Rost,
1995). Although, in general, epidermal and cortical cells
are treated as physiologically equal in many analyses of
nutrient uptake, they may not be, and the epidermis in
many situations will be quantitatively more important.
It has been generally assumed that absorbed nutrients
mostly move symplastically from epidermal cells through
the cortex to the stele; the Casparian Strip of the endodermis providing a major barrier for the apoplastic
movement of solutes and water into the stele. This barrier
means that to enter the stele, any nutrient ions reaching
the Casparian Strip must be taken up into endodermal
cells. Thus all nutrients bound for the xylem, whichever
route they follow to the endodermis, are in the symplast
Partitioning of transport in roots
at this point on the trans-root transfer pathway. To reach
the xylem these ions must subsequently be secreted
into the stelar apoplast. A corollary of this is that two
protein-mediated steps are necessary for the delivery of
solutes to the xylem one for uptake into the symplast
(whether at the epidermis, cortex or endodermis) and
one for release from stelar cells (Clarkson, 1993).
Electrophysiological studies con®rm the presence of
transport proteins in the plasma membrane of trichoblasts that could catalyse the uptake of some nutrients.
The presence of inwardly rectifying Kq channels in wheat
root hairs has been shown (Gassmann and Schroeder,
1994), while there has been strong evidence for the
presence of Hq-coupled nitrate in¯ux (Meharg and Blatt,
1995). The higher density of plasmodesmata at the
interface between the base of trichoblasts and the outer
layer of cortical cells (Vakhmistrov and Kurkova, 1979;
Vakhmistrov et al., 1981) is also consistent with a role
for root hairs in the uptake, then subsequent symplastic transfer, of nutrients into the plant. Similarly,
epidermal and cortical cells possess both low and high
af®nity transport systems capable of catalysing uptake
of a variety of different nutrients (Chrispeels et al., 1999;
Maathuis and Sanders, 1999).
The symplastic pathway of ion transfer from the soil to
the stele requires transport of ions from cell to cell via
plasmodesmata. Surprisingly, current knowledge of the
pathway is remarkably sparse considering it underpins
a widely-accepted hypothesis. Unlike leaves where maps
of plasmodesmatal density (so-called `plasmodesmograms') have provided important indications of the role
of symplastic and apoplastic pathways in phloem loading
(van Bel, 1993), no detailed information is available on
the extent of these connections in mature roots. This is
urgently needed, if only to demonstrate that the pathway
can potentially operate across the whole root. There is
also a need to determine the extent to which disruption of
the symplastic pathway affects uptake and transfer of ions
to the xylem. With the likelihood that plasmodesmatal
proteins will soon be identi®ed (Lee et al., 2000), it might
be possible to down-regulate these proteins and so disrupt plasmodesmatal formation or structure. The consequences for trans-root movement of ions and water
could then be investigated.
The role of the Casparian Strip in limiting apoplastic
movements of solutes has been mainly shown by
incubating plants in membrane-impermeant compounds,
and visualizing their build-up in the apoplast immediately
adjacent to the endodermis (Nagahashi et al., 1974;
Enstone and Peterson, 1992). Studies of the kinetics of
appearance of labelled Ca2q and Mg2q in the cortex and
stele of mycorrhizal spruce roots also supports the notion
that a major barrier to apoplastic movement to the stele
is located at the endodermis (Kuhn et al., 2000). However, there is evidence suggesting that the apoplastic
2q
451
in Arabidopsis roots is not limited
movement of Ca
by the Casparian Strip (White et al., 2000), signi®cantly
at odds with dogma. Yeo et al. have also suggested
that a major pathway for the entry of Naq into the xylem
of rice roots is via an apoplastic pathway (Yeo et al.,
1987). If these latter results prove to be more general
(e.g. for different solutes, species and growing conditions;
Schreiber et al., 1999), it will demand a signi®cant reconsideration of the view that membrane transport must
occur for nutrients to enter the stele.
Electrophysiological techniques have demonstrated the
presence of ion channels that are likely to be responsible
for the loading of solutes into the xylem. Patch clamping
of protoplasts isolated from the xylem parenchyma of
barley and the stele of Arabidopsis showed clearly the
presence of both cation and anion channels with characteristics consistent with a role in the loading of solutes
into the xylem (Wegner and Raschke, 1994; Wegner and
de Boer, 1997; Maathuis et al., 1998; KoÈhler and
Raschke, 2000). In a comparative study of Kq channels
in the cortex and stele of maize roots, the existence of
distinct channel types in the two cell types has been
shown (Roberts and Tester, 1995). The plasma membrane
of cortical cells was dominated by a Kq channel that
would favour Kq in¯ux into the cells (and thus root),
whereas the stelar cells were dominated by a Kq channel
favouring Kq ef¯ux into the apoplast (and thus xylem:
Fig. 2). Such clear-cut differences in channel activities
were not seen in Arabidopsis, where similar activities of
an outwardly-rectifying Kq channel was measured in
protoplasts from both the cortex and stele, while
inwardly-rectifying Kq channels were twice as frequent
in stelar cells compared with cortical cells (Maathuis
et al., 1998). However, given the clear effect of environment on these patterns of expression (see below), the
signi®cance of such observations is uncertain.
Underlining the likely physiological relevance of the
different channel types observed in the cortex and stele
of maize roots, Roberts found that addition of ABA to
intact plants for 12 h inhibited the stelar Kq outwardly
rectifying channel, but this treatment had no effect on the
cortical inwardly rectifying channel (Fig. 3) (Roberts,
1998). This mirrors results in intact plants where the
application of ABA rapidly inhibits the transfer of solutes
to the shoot, whilst having little effect on their uptake
into the root (Cram and Pitman, 1972). Furthermore,
expression of SKOR, a gene encoding a stelar outwardlyrectifying Kq channel, has been shown to be rapidly
inhibited by ABA (Fig. 4), whereas transcript from the
gene encoding, AKT1, the channel responsible for at least
some of the initial Kq uptake into the cortex (Hirsch et al.,
1998) is unaffected (Gaymard et al., 1998).
The results described above, at levels ranging from the
gene to the whole plant, illustrate clearly the importance
of cell-speci®c control of transport from the soil solution
452
Tester and Leigh
Fig. 2. Demonstration of a Kq in¯ux channel in cortical cells and a Kq ef¯ux channel in stelar cells of maize roots. Patch clamping was used to
measure Kq currents across the plasma membrane of isolated protoplasts (from Roberts and Tester, 1995, where further details can be obtained).
to the xylem. This highlights the need for a more targeted
approach for the genetic manipulation of nutrient uptake,
whether done for further investigation of underlying
processes or for plant improvement. Gene misexpression
must be in speci®c cell types, rather than constitutively
throughout the whole plant. Thus, either cell-speci®c promoters need to be catalogued and their wide availability
ensured, or other systems need to be developed for
manipulating gene expression in speci®c cells.
One such system has been developed in Arabidopsis
(see http:uuwww.plantsci.cam.ac.ukuHaseloffuHome.html).
In this, GAL4, which is a strong transcriptional activator
from yeast, has been modi®ed to provide high levels of
activation of genes harbouring the upstream activation
sequence (UAS) to which GAL4 binds (Brand and
Perrimon, 1993). This has been used to create `enhancer
trap' lines in Arabidopsis by random insertion into the
plant genome of a construct that contains the GAL4 gene
and a separate, UAS linked to a gene (mGFP5-ER) for
ER-targeted green ¯uorescent protein (GFP) (Fig. 5A;
J Haseloff, S Hodge, M Bauch, K Siemering, HM
Goodman, unpublished results; Kiegle et al., 2000b).
Insertion of this construct in fortuitous locations in the
plant genome places the GAL4 expression under the
control of endogenous plant promoters, resulting in its
cell-speci®c expression which can be visualized by the
presence of GFP resulting from the binding of GAL4 to
the UAS linked to the mGFP5-ER gene. Lines with the
cell-speci®c patterns of GFP expression can be selected
from a library of such enhancer-trapped plants. Using
this system, lines with cell-speci®c expression of GFP, and
hence GAL4, in the epidermis, the elongation zone
cortex, the endodermis, and the pericycle of the root
have been obtained (Fig. 6). These lines not only give
insights into the existence of interesting patterns of gene
expression in roots, but the GFP is a useful cell marker
that can be used to identify protoplasts derived from
particular cell types (Kiegle et al., 2000a). However, the
real power of the system is to use the presence of the
exogenous transcription factor, GAL4, to activate transcription of any other genes transformed into the
enhancer trap lines using a construct that contains the
gene of interest downstream of a second GAL4 UAS
(Fig. 5B). This leads to the activation of expression of the
introduced gene in the GFP-marked cells in which the
GAL4 gene product is also expressed.
In work to test the utility of this system to drive
targeted gene expression, transformation with a construct
Partitioning of transport in roots
453
Fig. 4. Down-regulation by ABA of transcription of a gene encoding
a stelar-speci®c outwardly rectifying Kq channel (SKOR), with no
effects on an inwardly rectifying Kq channel gene (AKT1). Northern
blot indicating transcript levels for the genes SKOR and AKT1 at
various times after pre-treatment of Arabidopsis with 10 mM ABA (from
Gaymard et al., 1998, where further details can be obtained).
Fig. 3. A Kq ef¯ux channel in stelar cells is speci®cally inhibited by
ABA. Mean current densities of whole cell inward and outward currents
in protoplasts isolated from the cortex and stele of maize roots that had
been well watered (WW), not watered for 60 h prior to protoplast
isolation (WS), or watered with 20 mM ABA 12 h before protoplast
isolation (ABA). Note the lack of inhibition by ABA of the inwardly
rectifying Kq currents in cortical protoplasts, but the large inhibition of
the outwardly rectifying currents in stelar protoplasts (from Roberts,
1998, where further details can be obtained).
Fig. 5. Constructs used for GAL4-UAS transactivation of genes. (A) An enhancer trap construct for random insertion into the plant genome.
Fortuitous expression of GAL4 driven by endogenous promoters at the point of insertion provides GAL4 protein which binds to the GAL4-UAS
in the second part of the construct. This drives the production of GFP from mGFP5-ER, allowing visualization of cell-speci®c patterns of expression.
(B) Construct for GAL4-dependent expression of a second gene (in this case a YFP-aequorin fusion). Introduction of this construct into the enhancer
trap lines leads to the expression of the gene, driven by the GAL4 produced by the previously-introduced construct shown in part A (see Fig. 6
for examples, for details, see Kiegle et al., 2000b).
454
Tester and Leigh
Fig. 6. Cell-speci®c expression of GAL4-mGFP5-ER and YFP-aequorin in Arabidopsis roots. (A) Cross-sectional diagram of an Arabidopsis root.
(B±E) Laser scanning confocal microscopic images of aequorin in different Arabidopsis lines. Cell walls were stained with propidium iodide
(red ¯uorescence). GFP and YFP are localised to (B) the epidermis, (C) the cortex of the elongation zone, (D) the endodermis, and (E) the pericycle.
(F, G) High magni®cation localization of GFP and YFP in epidermal cells of line J0481. Scale bars for B±E ˆ 50 mM; F ˆ 25 mM (for details see
Kiegle et al., 2000b).
containing a yellow ¯uorescent protein (YFP)-aequorin
fusion under the control of ®ve repeats of the GAL4 UAS
(Fig. 5B) resulted in expression of YFP-aequorin in cells
labelled with GFP (Fig. 6). These plants have been used
to investigate the effects of stress on cytosolic Ca2q
activities in various cell types (Kiegle et al., 2000b).
Cell-speci®c expression of aequorin revealed cell-speci®c
alterations in cytosolic Ca2q in response to NaCl or water
stress, including clear oscillations that were not evident
in plants expressing aequorin constitutively (Fig. 7).
This system can be clearly used not only to monitor
plant function, but also to manipulate it. Due to the
Partitioning of transport in roots
455
when combined with the use of microarrays, will enable
rapid identi®cation of cell-speci®c expression patterns.
It is expected that, with these new technologies and
perspectives, the coming years will bring many advances
to the understanding of root function.
Supplementary Material
Supplementary material is available at JXB Online.
References
Fig. 7. Representative measurements of light emission from aequorin
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studies of the control of nutrient uptake. For example,
the relative role of the cortex versus epidermis in nutrient
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Conclusions
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