Journal of Experimental Botany, Vol. 47, No. 300, pp. 843-854, July 1996
Journal of
Experimental
Botany
REVIEW ARTICLE
Nitrate transport and compartmentation in
cereal root cells
Anthony J. Miller1 and Susan J. Smith
Biochemistry and Physiology Department, lACR-Rothamsted, Harpenden, Herts. AL52JQ, UK
Received 3 September 1995; Accepted 5 February 1996
Abstract
Measurement of cytosolic nitrate is one of the factors
required for the resolution of factors controlling nitrate
uptake and assimilation in plants and for identifying
likely nitrate transport mechanisms at both the plasma
membrane and tonoplast. This paper reviews methods
and reported measurements of cytosolic nitrate in
higher plants and concludes that nitrate-selective
microelectrodes are the best approach. These microelectrodes have been used to measure intracellular
nitrate activitites in barley and maize root cells. Triplebarrelled electrodes, incorporating a pH-sensing barrel
have been used to identify the compartmental location
of the nitrate-selective tip giving unequivocal estimates of vacuolar and cytosolic nitrate activities. The
microelectrode measurements are used to discuss the
possible mechanisms of nitrate transport at both the
tonoplast and plasma membrane. The energetics of
possible proton-coupled transport systems are
described and the feasibility of the mechanism is
discussed.
Key words: Cytosol, compartmentation, Hordeum vulgare
L, nitrate, roots, Zea mays L
requisite to understanding the regulation of nitrate
transport. Furthermore, cytosolic nitrate concentration
must influence nitrate reductase (NR) activity and could,
therefore, determine the rate of nitrogen assimilation. The
methods that have been used to measure intracellular
nitrate have been reviewed here with particular emphasis
on nitrate-selective microelectrodes. The accuracy of each
method is assessed, and the results obtained with microelectrodes are used to appraise possible nitrate transport
mechanisms at both the plasma membrane and tonoplast.
Methods for measuring intracellular nitrate
Several techniques have been used to try to measure
intracellular nitrate concentration, including estimates
based on NR activity, compartmental radiotracer efflux
analysis, nuclear magnetic reasonance (NMR) and cell
fractionation. Particular emphasis has been placed on
trying to estimate cytosolic nitrate concentrations because
of its metabolic importance but the various techniques
have yielded different values of this parameter (Table 1).
This spread of values suggests that either cytosolic nitrate
is very variable or there are errors associated with each
of the methods used to measure it.
Anaerobic NR assay
Introduction
The measurement of cytosolic and vacuolar nitrate concentration is essential for determining the mechanism of
nitrate transport at both the plasma membrane and
tonoplast because the electrochemical gradient of this ion
across each of these membranes determines whether the
uptake of nitrate into the cell and into the vacuole occurs
by passive or active mechanisms. Clarkson (1986) concluded that measurements of cytosolic nitrate are a pre1
The anaerobic NR method has been used to estimate the
'metabolic' nitrate pool and is based on measurement of
the nitrite formation in the absence of external nitrate,
under conditions (anoxia, darkness) intended to inhibit
nitrite reduction (Ferrari et cil., 1973). This technique
depends on the cytosolic location of NR, and the resulting
limited access of the enzyme to its substrate, nitrate. The
cessation of nitrite formation is regarded as the indication
of exhaustion of the 'metabolic' nitrate pool which is
To whom correspondence should be addressed. Fax: +44 1582 760981. E-mail tony.miller©bbsrc.ac.uk
Abbreviations: JG'/F, free-energy change for H+/NOj~-symport; F, Faraday constant; pHc, cytoplasmic pH; pHo, external pH; pINOJ,,, -log10 cytosolic
NO^" concentration; pmf, proton motive force; pINOJo, -tog10 externaJ NO^~ concentration; NR, nitrate reductase; NMR, nuclear magnetic resonance.
© Oxford University Press 1996
844 Miller and Smith
Table 1. Higher plant cytosolic nitrate concentrations estimated by a variety of techniques
Method
Tissue
Nitrate concentrations (mol m
Solution
Anaerobic nitrate
reductase assay
Compartmental tracer
efflux analysis
Cell fractionation
Barley, maize, pea,
soybean, nee leaves
Spinach leaf
Barley root
Barley root
Maize root
Barley root
Barley root
Onion root
Soybean root
Barley leaf
Mesophyll cell
4.0
0.1-20
0.01
1.4-70
1.5
0.01-1
2.0
0.5
assumed to be synonomous with cytosolic nitrate.
Cytosolic nitrate values obtained using this type of
approach range from 0.01 to 8 mol m" 3 (Table 1).
One problem with this method is that nitrite formation
during the analysis may be controlled by the distribution
and activity of the nitrate reductase (NR), the supply of
reductant, as well as the availability of nitrate itself
(Hageman et al., 1980). Furthermore, under the completely anoxic conditions required, and as cytosolic nitrite
accumulates, the intracellular distribution of nitrate, particularly its release from the vacuole, may be disturbed,
resulting in a non-physiological situation. For example,
under these anoxic conditions it has been shown that
stored nitrate leaks into the external solution (Aslam,
1981). The method has been modified to estimate in vivo
rates of NR activity under anaerobic conditions and from
the Michaelis-Menten kinetics to calculate [NO3~]C, the
approach assumes that NR activity in vivo must be limited
by available nitrate (Robin et al., 1983; King et al., 1992).
These authors argue that because the addition of more
(up to 100 mol m~3) nitrate outside the root enhanced
the anaerobic NO^~ production, NR activity in vivo must
be limited by the supply of nitrate rather than by supply
of reductant or some other factors. Nevertheless, the
main disadvantage of this technique must be that estimates of cytosolic nitrate are made under anaerobic conditions, a situation which may in itself alter the [NO3~]C.
Kaiser and Huber (1994) have reviewed how several
processes, including oxygen availability, can rapidly
modify the in vivo activity of NR. However, one advantage
of this method is that the final calculation of cytosolic
nitrate by this method does not require any assumptions
of cytosolic volume in the tissue (cf. below).
Compartmental tracer efflux
Compartmental tracer efflux analysis is a well-established
technique and has been widely used for estimating fluxes
and subcellular compartmental concentrations of a variety
3
)
References
Cytosol
0.01-0.1
Robin et al. (1983)
4-8
0.66-3.9
82
SteingrSver et al. (1986)
King et al. (1992)
Deane-Drummond
and Glass (1982)
Presland and McNaughton (1984)
Lee and Clarkson (1986)
Siddiqi et al. (1991)
Macklon et al. (1990)
Mullere(a/ (1995)
Martinoia el al. (1986)
Martinoia el al. (1987)
6-160
26
12-37
40-50
4-8
4.1
6.8
of ions (MacRobbie, 1971). For this method, tissues are
loaded to a steady-state (constant specific activity in all
compartments) with an isotopic tracer and then put in an
unlabelled solution (efflux solution) of the same composition as the loading solution. The efflux solution is replaced
periodically and its tracer content determined. The plasma
membrane and tonoplast offer different resistances to
tracer efflux and the kinetics of tracer loss from the tissue
into the solution can be analysed in terms of several
exponential components each corresponding to different
tissue compartments. This analysis yields values for
steady-statefluxesacross the plasma membrane and tonoplast as well as tracer content of the cytoplasm and
vacuole, which can be converted to concentration if the
compartment volumes are known.
Presland and McNaughton (1984), using 13NOf,
estimated cytosolic concentrations of nitrate from 6 to
160 mol m~3 in maize roots over external nitrate concentrations of 1.4 to 70 mol m~3. Using the same tracer, Lee
and Clarkson (1986) estimated cytosolic nitrate concentrations of 26 mol m" 3 in barley root cells growing in 1.5
mol m~3 nitrate. Siddiqi et al. (1991), also using 13NOf,
reported that in barley roots cytosolic nitrate concentration increased from 12 to 37 mol m~3 when external
nitrate increased from 0.01 to 1 mol m~3. In contrast,
with 15NO3~, Macklon et al. (1990) reported a cytosolic
nitrate concentration of 40-50 mol m~3 in onion roots.
Measurements with the nitrate analogue, 36C1O3, by
Deane-Drummond and Glass (1982) suggested a value
of 8 mol m~3 for cytosolic nitrate concentration in barley
roots grown in 0.01 mol m~3 nitrate. The differences
between these estimates of cytosolic nitrate concentrations
could be partly due to the differences in the pretreatment
given to the cells, in the experimental procedures used,
or genuine species differences.
The radioisotopes of nitrogen are not very convenient
for this type of analysis, 13N has a very short half-life
(ti 2 = 9.9 min) and is not readily available. While for the
Nitrate transport and compartmentation in cereal roots
14
stable isotope N, it may be difficult to obtain sufficient
enrichment of activity, relative to the naturally occurring
15
N, in efflux washes. The activity of 15N to 14N in the
efflux sample is important because the isotopic measurement techniques are relatively insensitive, although
MackJon et al. (1990) successfully used this isotope for
onion roots. However, recent improvements in the sensitivity of mass spectrometers suggests that there may be
greater potential for the use of 15N (Muller et al., 1995).
The physiological consequences of using 36C1O3 as an
analogue for nitrate in compartmental and transport
studies have been questioned because chlorate can be
readily reduced to toxic chlorite by NR in plants (Murphy
et al., 1985). Another problem with compartmental efflux
analysis is that the labelled nitrate present in the cytosol
is continuously being assimilated and also being transported to the xylem and so is no longer available for
efflux. Nitrate taken up by roots can be found in the
shoots within a few minutes (McNaughton and Presland,
1983), this makes it difficult to analyse tracer efflux
kinetics from subcellular compartments and, therefore, to
estimate intracellular nitrate concentrations correctly. The
removal of nitrate from the cell can result in continuous
changes in specific activity of nitrate pools although
authors have attempted to correct for this problem (Lee
and Clarkson, 1986).
Nuclear magnetic reasonance
There has been one report of the application of 14N
NMR to the measurement of intracellular nitrate compartmentation, and a single large pool of nitrate was
detected (Belton et al., 1985). The concentration of nitrate
in this pool was very close to that determined by extraction of nitrate from the whole tissue and this was,
therefore, taken to be the vacuolar nitrate pool. NMR
has the advantage of being relatively rapid and nondestructive, but is of low resolution and has a relatively
poor signal:noise ratio so its use in detecting nitrate in
tissues with low concentrations of the ion or for measuring
the relatively small pool of cytosolic nitrate is limited.
Also, there are no easy methods for separating signals
from the cytosol and the vacuole in contrast to 31 P for
which differences in the pH of these compartments separates the signal from each (Lee et al., 1990). Therefore,
NMR probably has limited utility in detailed studies of
intracellular nitrate pools.
Cell fractionation
Vacuole isolation from protoplasts has been used to
estimate nitrate in barley leaf vacuoles (Martinoia et al.,
1981; Granstedt and Huffaker, 1982) and nitrate distribution between the cytosol and vacuole (Martinoia et al.,
1986, 1987). Analysis of vacuoles isolated from barley
leaves demonstrated that between 58% and 99% of proto-
845
plast nitrate is present in the vacuole (Martinoia et al.,
1981; Granstedt and Huffaker, 1982). Using the same
procedure, Martinoia et al. (1986, 1987) measured nitrate
concentrations in the range of 4 to 7 mol m~ 3 in the
extravacuolar space of barley mesophyll protoplasts (presumably largely representative of the cytoplasm). A
freeze-fractionation procedure (Gerhardt and Heldt,
1984) has also been used to estimate subcellular concentrations of metabolite levels, including nitrate in leaves
of barley (Winter et al., 1993) and spinach (Winter
etai, 1994).
Vacuole isolation requires a lengthy preparatory procedure to obtain protoplasts and vacuoles and there is
the possibility for solute leakage or redistribution during
the preparation, which may result in calculated cytosolic
nitrate concentrations which do not reflect those in vivo.
The value calculated for the extravacuolar concentration
is the average for the whole cytoplasm including all
organelles, not just the concentration in the cytosol.
Most of the above techniques measure the amount of
nitrate in different subcellular pools and then convert this
to a concentration in different compartments using an
estimate of the volume of the compartment. Although
the size of this volume estimate can be based on quantitative microscopy, relatively small errors can lead to large
differences in calculated concentrations. For instance, the
calculated nitrate concentration increases 2-fold when the
assumed cytosolic volume changes from 10% to 5% of
the total cell volume. In addition, protoplasts and vacuoles are usually prepared in hypertonic media that will
change the volumes of compartments from those in vivo.
Although this can be compensated for (Leigh et al.,
1981), this is not usually done and so the estimated
nitrate concentration will not reflect those in the original tissue.
In order to explain some of the differences in the
apparent cytosolic nitrate concentrations of barley roots
obtained by these different methods (Table 1), two different cytosolic pools of nitrate have been proposed to be
present in roots (the tissue most often used for the
estimations), a large slowly metabolized pool, possibly
the cytoplasm of cortical cells, and a smaller
NR-containing pool, possibly the cytoplasm of epidermal
cells (Siddiqi et al., 1991; King et al., 1992). The tissue
heterogeneity found in vivo NR localization studies may
provide some support for this idea (Rufty et al., 1986;
Fedorova et al., 1994). However, all of the above techniques average the compartmental nitrate for the whole
root, none samples single cells.
Nitrate-selective microelectrodes
A more direct approach to measuring cytosolic nitrate is
to use nitrate-selective microelectrodes (Miller and Zhen,
1991). Ion-selective microelectrodes all have the same
846 Miller and Smith
basic design, with a hydrophobic ion-selective sensor
plugging the tip of a glass micropipette; the ion-dependent
electrical potential is measured across this barrier (Miller,
1994). For intracellular measurements it is necessary to
use double-barrelled microelectrodes in which one barrel
contains the ion-selective sensor and the other measures
the cell's membrane potential. This is necessary because
when inserted into the cell, the ion-selective sensor gives
an output that is the summation of its response to the
local ion concentration and the membrane potential. The
latter must be subtracted to give the ion-dependent
response of the sensor. A major advantage of electrode
measurements is that there is no need for compartmental
volume assumptions because the electrodes directly report
ion activity, the thermodynamically important parameter.
In addition, microelectrodes also measure the membrane
electrical potential difference and this information,
together with the ion activities can then be used to
evaluate the possible mechanisms of transport across the
membranes. Also, the cell membrane potential indicates
the health of the cell during the impalement because when
the plasma membrane is damaged the cell is unable to
maintain a stable resting membrane potential.
There are two reports of microelectrode measurements
of cytosolic nitrate in lower plants, 1.6 mol m~3 for
Chara corallina (Miller and Zhen, 1991) and 0.63 mol
m" 3 for thallus cells of the liverwort, Conocephalum
conicwn (Trebacz et al., 1994). Electrodes have also been
used to measure both the cytosolic and vacuolar nitrate
activities of barley root epidermal cells growing in a full
nutrient solution containing 10 mol m" 3 nitrate (Zhen
et al., 1991; Miller and Smith, 1992). A comparison of
measurements of vacuolar and cytosolic nitrate concentrations in barley and maize roots obtained using doublebarrelled nitrate-selective microelectrodes is shown in
Table 2. When using double-barrelled electrodes in tissues
grown at relatively high external concentrations (10 mol
m~ 3 ), the values obtained fall into two groups (data for
maize roots in Fig. 2; see Zhen et al., 1991, for similar
results on barley roots). Using a single cell sampling
technique, Zhen et al. (1991) showed that the population
with the larger nitrate concentration was vacuolar in
origin and, therefore, by implication, the one with the
smaller concentration was cytosolic. The mean cytosolic
activity in barley was 4.9 mol m~3 and that for maize
was 3.1 mol m~3. Corresponding vacuolar nitrate activities were 39 and 26 mol m~3, respectively (Table 2).
Although it was relatively easy to assign activities to
the vacuole and cytosol in roots growing in solution with
10 mol m~3 nitrate, this was less easy when plants were
grown in low external nitrate concentration (e.g. <0.1
mol m~ 3 ). Under these conditions, the nitrate activity in
the vacuole is low and measurements can no longer be
separated into two populations. This problem can be
overcome by using triple-barrelled microelectrodes
12
10 -
o
6 H
Urn
4)
i
4
~
2 -
0-2
2-4
4-6
10-20 20-30 30-40 40-50 50-60
NO3~activity (mol m"-3\)
Fig. 1. Histogram showing the distribution of nitrate activities measured
with nitrate-selective microelectrodes in epidermal cells of maize roots
grown for 24-30 h in a full nutrient solution containing 10 mol m~ 3
nitrate. A total of 43 measurements separated into two populations
with means (with 95% confidence limits) of 3.1 (2.7, 3.5) and 26.2
(24, 28.6).
incorporating a pH sensor in the third barrel (Walker
et al., 1995). These allow unequivocal identification of
the cytosol and vacuole from their pH values: approximately 7.4 and 5.5, respectively (Kurkdjian and Guern,
1989). Triple-barrelled microelectrode measurements for
barley root cells growing in full nutrient solution at pH 6
containing a range of different nitrate concentrations,
from 10 to 0.1 mol m~3 show that cytosolic nitrate is
maintained at around 4 mol m~3 (Miller and Smith,
unpublished results).
Mechanisms of nitrate transport at the plasma
membrane
As indicated above, an advantage of using nitrate-selective
microelectrodes is that they give values for membrane
potentials and compartmental nitrate activities that can
be used to estimate the thermodynamic or free energy
gradient for nitrate transport across the plasma membrane
and tonoplast. For example, insertion of the values for
barley and maize cytosolic nitrate concentrations and
electrical potential across the plasma membrane into the
Nernst equation indicates that the cytosolic nitrate concentration is greater than can be achieved by a passive
transport process even at a high external nitrate concentration (Zhen et al, 1991). A passive transport mechanism could only maintain micromolar concentrations of
nitrate in the cytosol.
Nitrate transport and compartmentation in cereal roots
847
Table 2. A comparison of mean (with 95% confidence limits) cytosolic and vacuolar nitrate activities in barley fHordeum vulgare L.
cv. Klaxon) and maize (Zea mays L. cv. Eta) root epidermal cells determined using double-barrelled nitrate-selective microelectrodes
Results (mean±s.e.) from chemical analysis of the whole root tissue are also presented for comparison. Plants were grown in 10 mol m~ 3 nitrate
for 24-30 h under 16 h daylength. Whole-root nitrate was extracted and measured as described by Zhen el al. (1991). All measurements were made
between 1-2 cm from the root tip. The epidermal cells were identified as the first layer of cells encountered by the microelectrode.
Plant
Barley
Maize
Cytosol
Tissue nitrate
(mol m~ 3 )
Vacuole
Nitrate
(mol m~ 3 )
Membrane potential
(mV)
Number of
measurements
Nitrate
(mol m~ 3 )
Membrane potential
(mV)
Number of
measurements
4.9(4.5, 5.5)
3.1(2.7,3.5)
-73±6
-63±5
19
12
39(37,42)
26 (24, 27)
-65±4
-66 + 3
35
31
75±6
50±4
Passive nitrate transport
1:1 H7NO 3 symport
-
100 -
9
f
0 -
i
•
— —'
_—
I
l
100 - 5 '
i
PH0
8
200 300 /inn
2:1 H + /NO 3 ' symport
200
3:1 H 7NO 3 symport
onn —,
100 0 -
9
i
1
-100 - 5
6
pHc
-200 -300 -400 -500 .Ann
Fig. 2. Calculation of AGjF for a plasma membrane symport mechanism with either a 1.1, 2:1 or 3. I H + NO3~ stoichiometry. Three lines
are shown for each stoichiometry, these represent three different external
nitrate concentrations: 0.1 (
), 1 (
) and 10 (
) mol m" 3 .
Values used for these calculations are based on those obtained from
measurements in barley root epidermal cells: plasma membrane potential
(/) V) is - 7 0 mV, pH c is 7.2 and pINOj], is 2.4 (i.e. 4 mol m~ 3 ). These
values are assumed to be maintained independently of changes in
external pH and nitrate concentration. A positive free-energy value
indicates that this mechanism could not maintain the observed nitrate
gradients across the plasma membrane.
Passive nitrate movements across membranes are likely
to be via ion channels; a nitrate-permeable channel, which
would allow the flow of anions into the cell, has been
identified in the plasma membrane of wheat protoplasts
(Skerrett and Tyerman, 1994). Such a channel may have
a role in the 'constitutive' uptake system when nitrate is
first supplied (Behl et al., 1988). Subsequently, an active
nitrate uptake system is induced or derepressed by the
presence of cytosolic nitrate. Even though passive uptake
could only produce micromolar nitrate concentrations in
the cytosol, this may be sufficient for 'induction' of nitrate
transport and assimilation, without the need for a nitrate
receptor on the outside of the cell as discussed by
Redinbaugh and Campbell (1991). Alternatively, the
channel itself could be a receptor, with binding of the
nitrate ion to the channel as the signal for induction.
An anion channel could also provide the mechanism
for nitrate efflux which has been reported by many
authors (Jackson et al., 1986, and references therein). The
direction of flow of anions through a channel is determined by the electrochemical gradient for the ion, but
rectification (one-way movement of current) of the channel will determine if it has a specific role as an influx or
efflux mechanism. For example, the anion channel
described by Skerret and Tyerman (1994) in wheat root
protoplasts only allows the passage of anions into the cell
(anion outward rectifier) and so could not be a mechanism
for nitrate efflux. By contrast, the stretch-activated channel of tobacco protoplasts (Falke et al., 1988) and the
voltage-regulated channels of guard cells (Schmidt and
Schroeder, 1994) allow efflux of anions. The regulation
of plasma membrane anion channel activity may be
important in determining cytosolic nitrate concentrations,
because as active transport is maintaining the cytosolic
nitrate a large 'leak' through a channel will quickly
deplete cytosolic nitrate. An open channel has selective
permeability allowing some ions to flow passively down
their electrochemical gradient at a great rate (106— 108
ions s" 1 ; Sanders and Slayman, 1989). Assuming a cytosolic volume of between 2 and 10 pi per cell (1-5% of
the whole cell volume; Malone et al., 1991) and no active
848
Miller and Smith
influx of nitrate, a single open anion channel with an
efflux rate of 107 ions s~' will deplete the cytosolic nitrate
concentration from 4 mol m~3 to mmol m~3 levels (passive nitrate distribution) in 50-200 s.
Active nitrate transport
Active transport is required at the plasma membrane and
the tonoplast of epidermal cells in both maize and barley
roots to maintain the measured intracellular concentrations of nitrate. The proton electrochemical gradient
across both the tonoplast and the plasma membrane can
provide the energy for the transport of nitrate. Active
nitrate transport at the plasma membrane is thought to
occur by symport with protons. Measurements in maize
and barley of the nitrate-elicited changes in electrical
potential difference across the plasma membrane support
a proton symport model (McClure et al., 1990; Glass
et al., 1992). These measurements suggest that the symport must have a stoichiometry of at least 2:1 H + :NO3"
as 1 :1 would be electrically neutral and would not cause
depolarization of membrane potential. Nitrate uptake
studies have identified two distinct phases of nitrate
uptake which are dependent on the external concentrations of nitrate, these are described as high affinity and
low affinity uptake (Doddema and Telkamp, 1979). For
barley roots, only the high affinity system showed hyperbolic kinetics which saturated at external nitrate concentrations between 0.2-0.5 mol m~3, while the low affinity
system did not saturate over the external concentration
range 0.5-50 mol m~3 (Siddiqi et al., 1990). The latter
authors suggested that such linear kinetics are consistent
with a channel uptake mechanism. However, Glass et al.
(1992), by measuring nitrate-elicited changes in membrane potential, showed that in barley roots both high
and low affinity nitrate uptake systems appear to be 2:1
H + :NO 3 ~ symport mechanisms. Furthermore, a lowaffinity nitrate transporter from Arabidopsis has been
characterized as having a proton:nitrate stoichiometry of
2:1 (Tsay et al., 1993).
The thermodynamic feasibility of a proton symport
mechanism over both high and low affinity uptake ranges
can be determined by using the measurements of cytosolic
nitrate activities, pH and membrane potentials obtained
with triple-barrelled nitrate-selective microelectrodes. For
a H+/NO3~ symport at the plasma membrane, the appropriate free-energy relationship for the reaction is
+ (n-\)AW
(1)
where n is the stoichiometry of protons to nitrate ions
for the symport, and A *¥ is the trans-plasma membrane
potential difference and subscripts o and c denote the
external solution and cytosol, respectively. The free energy
for the symport is expressed numerically in mV. The free
energy required to maintain a cytosolic nitrate concentration of 4 mol m~3 can thus be calculated for different
values of n. By calculating the free energy at different
external nitrate and pH values, the ability of different
symport mechanisms to maintain cytosolic nitrate can be
assessed. The results of such calculations are shown
plotted in Fig. 2. For the calculations it was assumed that
the cytosolic nitrate concentration remained at 4 mol m" 3
at all external nitrate concentrations (see above), that the
cytosolic pH was 7.2 (Miller and Smith, 1992) and was
insensitive to external changes pH (see Kurkdjian and
Guern, 1989, and references therein), and that the membrane potential was -70 mV (Zhen et al., 1991). Although
it remains to be established that all parameters would
remain constant under the combination of conditions
assumed, the calculations give an estimate of the ability
of the different mechanisms to account for the observed
cytosolic nitrate concentration.
Thermodynamics of low and high affinity nitrate transport at
the plasma membrane
Does proton symport require a 2:1 stoichiometry in the
low affinity uptake range? The free energy values plotted
in Fig. 2 show how increasing the stoichiometry value n
increases the slope of the graph. When n=\ the free
energy values are smaller than for higher n values indicating that the transport mechanism will be more sensitive
to the pHo. However, it is feasible for low-affinity nitrate
symport to have this stoichiometry, at external concentrations above 0.5 mol m" 3 nitrate and below a pHo of 6.5
(Fig. 2). The main disadvantage of an electroneutral 1:1
cotransport is that it is energized by only the pH gradient
across the plasma membrane; the A V term disappears in
equation 1. Therefore, at any given external nitrate concentration, such a mechanism would be totally dependent
on the pH gradient across the plasma membrane, a
parameter which it is difficult for the cell to adjust in
response to the prevailing environmental conditions. The
pH-buffering capacity of the cell wall and the cytosol,
together with the necessary pH regulation of the cytosol
for the biochemical processes results in very little flexibility for a plant cell in terms of adjusting the plasma
membrane pH gradients in order to maintain nitrate
uptake in response to changes in external nitrate concentration. The fundamental importance of nitrate as a
nutrient ion surely requires that uptake could not be
powered by such an unreliable and inflexible energy
source.
In contrast to a n — 1 stoichiometry, Fig. 2 indicates
that a 2:1 proton:nitrate symport would support nitrate
transport in all circumstances except when the external
concentration of nitrate is 10 mol m~3 and the external
pH is 8 (few agricultural soils are likely to be more
alkaline than this). Only, at lower external nitrate concen-
Nitrate transport and compartmentation in cereal roots 849
trations does this situation become acute, with a pH o of
7 becoming limiting at 0.1 mol m~ 3 external nitrate. For
2:1 symport the membrane potential becomes important
for nitrate uptake as it can ensure that there is sufficient
energy for transport. Comparing the graphs of 2:1 and
3:1 stoichiometries in Fig. 2 there is little advantage in a
mechanism with a 3:1 stoichiometry, i.e. there is little
difference in the positive AG/F area on the two graphs,
indicating that there is little energetic gain obtained by
increasing the proton to nitrate stoichiometry to the
unlikely value of 3. Such a high stoichiometry would be
unusual but not impossible. In Fig. 2 for both 2:1 and
3:1 stoichiometries, as the plasma membrane potential
becomes more negative so the energetic profile shifts
down along the y-axis to negative AG/F values and
symport becomes feasible.
The importance of the plasma membrane voltage in
supplying energy for nitrate uptake is shown graphically
in Fig. 3. This figure shows the plasma membrane potential difference which is required for a 2:1 stoichiometry
to maintain the cytosolic nitrate concentration at 4 mol
m~ 3 at a range of different external pH values and with
different external nitrate concentrations. For example, at
an external pH of 7.5 and a nitrate concentration of 0.01
mol m~ 3 a membrane potential of —200 mV is required
for a 2:1 symport mechanism to maintain cytosolic nitrate
at 4 mol m " 3 the requirement for any membrane potential
Plasma membrane potential difference
required for 2H+/NOj symport
-200
PH O
Fig. 3. A graph showing the plasma membrane potential difference
which is required for a 2:1 H + : NOf symport to maintain cytosolic
nitrate at 4 mol m " 3 at a range of external pH values and at different
external nitrate concentrations.
component of the proton motive force (pmf) disappears
at this external nitrate concentration when the external
pH is <5.8. Thus both external pH and cell membrane
potential are important parameters for determining the
uptake of nitrate. Meharg and Blatt (1995) have described
a kinetic model for a 2:1 proton:nitrate symport in
Ambidopsis root hairs, their model also emphasizes the
importance of membrane voltage in controlling nitrate
transport.
Nitrate-elicited changes in membrane potential can be
regarded as an assay for nitrate symport activity. McClure
et al. (1990) found that in maize root cells at pH o 8,
when the resting membrane potential was —184 mV, the
nitrate-elicited (0.1 mol m~ 3 ) depolarization of membrane
potential disappeared. This result is consistent with the
thermodynamic calculations in Fig. 3 and is consistent
with the idea that under these conditions of pH o and A f
symport can no longer occur by a 2:1 mechanism because
they are close to the thermodynamic limits for this
stoichiometry (Fig. 2). In contrast, Ullrich and Novacky
(1981) in fronds of Lemna, at an external pH of 8.3 when
AT was —234 mV, observed a nitrate-elicited (2 mol
m~ 3 ) depolarization. This result too is consistent with
the thermodynamic profiles in Fig. 3 as the A G/F value
is negative (below the x axis in Fig. 2) under these
conditions because of the large negative A W.
The reported effects of external pH on net nitrate
uptake is variable and seems to depend on the species of
plant, for example, more acid optima for barley (Rao
and Rains, 1976) and more alkaline for Arabidopsis
(Doddema and Telkamp, 1979). However, some of the
variation may be explained by an apoplastic pH gradient
in some solutions (Grignon and Sentenac, 1991). In
barley, net nitrate uptake was measured by Aslam et al.
(1995). They found that a decrease in external pH from
5 to 3 decreased uptake, but concluded that this change
was due to an increase in efflux rather than a change in
influx. As net uptake reports the steady-state resulting
from efflux and influx of nitrate, the differing effects of
pH on uptake in different species may result from changes
in efflux rather than influx.
In the soil, barley and maize are likely to utilize both
nitrate and reduced forms of nitrogen, such as ammonium. One interesting consequence of using nitrate as a
nitrogen source is the associated alkalinization of the
surrounding medium (Raven and Smith, 1976). However,
the extent of any pH change occurring in the field will
depend on the pH buffering capacity of the soil. An
increase in pH at the surface of the root will not favour
a proton symport mechanism of nitrate uptake, perhaps
indicating that energy sources other than proton gradients
may also be needed at high external pH. Some authors
have reported that as external pH increases there is a
corresponding decrease in net nitrate uptake while ammonium uptake increases (Barber, 1984, and references
850 Miller and Smith
therein). Nitrogen uptake is so important for the growth
of any plant that it would seem reasonable to have several
different mechanisms available for uptake. Other possible
energy sources are sodium gradients and sodium-coupled
nitrate transport is known to occur in cyanobacteria
(Lara et cil., 1993) and various Na +-coupled transport
systems have been identified in giant algal cells (Walker
et al., 1993). It has recently been demonstrated that
sodium-coupled transport mechanisms can be driven by
proton gradients (Hirayama et al., 1994), so perhaps
NO3" cotransport can utilize different cation gradients.
One advantage for the plant cell in cotransporting nitrate
and Na + is that the entry of protons is avoided so
circumventing the pH problems reviewed by Raven and
Smith (1976). However, sodium cotransport introduces
its own problems as the cell can not tolerate large
accumulations of this ion in the cytosol, for example,
protein synthesis is sensitive to Na + concentration
(Gibson et al., 1984).
Another possible mechanism is to couple nitrate transport directly to the hydrolysis of ATP, but a nitrateATPase seems unlikely as it should have been identified
in plasma membrane vesicle studies, such as that by RuizCristin and Briskin (1991). Antiport of nitrate with
bicarbonate has been proposed (Imsande and Touraine,
1994), but there is no evidence for this mechanism.
Bicarbonate-coupled cotransport mechanisms do exist
(Zhao et al., 1995), but it is difficult to distinguish
between proton symport, and HCO^" or OH~ antiport.
Mechanisms of transport at the tonoplast
Triple-barrelled electrode measurements have shown that
the populations of measurements obtained using doublebarrelled electrodes may have tended to overestimate
mean vacuolar concentrations because some very low
vacuolar measurements had been assumed to be cytosolic
(Miller et al., 1995). Nonetheless, over the range of
external nitrate concentration from 1-10 mol m~3 an
active transport mechanism is required at the tonoplast.
Using the Nernst equation with a small trans-tonoplast
potential difference of 10-20 mV, passive transport across
the tonoplast could produce equilibrium vacuolar concentrations of 6-9 mol m~3 nitrate in the vacuole. As much
higher vacuolar concentrations are often found (e.g.
Fig. 1) active transport into the vacuole must occur.
Proton antiport mechanisms (Schumaker and Sze, 1987)
have been proposed and the thermodynamics of such
systems have been calculated (Miller and Smith, 1992).
These systems should be very electrogenic, as there will
be the net movement of two units of charge, the anion
NO^" into the vacuole in exchange for a H + out; such a
mechanism should be detected by whole-cell voltageclamp of isolated vacuoles. Experiments purporting to
show H + : NO^~ symport mechanisms in isolated tono-
plast vesicles (Blumwald and Poole, 1985) have been
shown to be an artefact resulting from the use of acridine
orange as a pH probe (Pope and Leigh, 1988).
Experiments using tonoplast vesicles have failed to
identify clearly an anion cotransport uptake mechanism.
This may be because these experiments did not use plants
previously grown in nitrate, and perhaps the tonoplast
transporter is nitrate-inducible. The plant cell will require
the active accumulation of nitrate only when the anion is
available and McClure et al. (1987) and Ni and Beevers
(1994) have identified nitrate-inducible proteins appearing
in the tonoplast and/or endoplasmic reticulum of maize
root cells. Isolated tonoplast vesicles have a large anion
conductance (Pope and Leigh, 1987) and this property
may dominate and so hide active nitrate uptake systems.
This anion channel activity in the tonoplast may be
involved in the remobilization of vacuolar stored nitrate,
but during vesicle isolation the regulation of channel
activity could be lost. Patch-clamp studies of isolated
vacuoles have also identified anion channel activity at the
tonoplast (reviewed by Tyerman, 1992).
Regulation of cytosolic nitrate
One of the main findings from using triple-barrelled
microelectrodes is that cytosolic nitrate activity is maintained constant over a range of external nitrate concentrations from 0.1-10 mol m" 3 (Miller et al., 1995*). Thus,
in common with other major nutrients such as K+ (Miller
et al., 1995a), POJ" (Lee et al., 1990) and also H +
(Kurkdjian and Guern, 1989) and Ca 2+ (Sanders et al.,
1990), cytosolic nitrate is probably regulated within relatively narrow limits.
Why regulate cytosolic nitrate?
Xylem loading of nitrate may be a passive process involving anion channels like those described in the xylem
parenchyma of barley roots (Wegner and Raschke, 1994).
Assuming the xylem parenchyma cells have similar cytosolic nitrate concentrations to those in epidermal and
cortical cells, xylem loading can be down the electrochemical gradient via channels. Indeed this may be the chief
reason for the plant cell to use energy to maintain
cytosolic nitrate at around 4 mol m~3. Unfortunately, it
is difficult to make an electrode impalement into a xylem
parenchyma cell in an intact root because it causes too
much damage to other adjoining cells. Another assumption is that the membrane potential of xylem parenchyma
cells and hence the electrochemical gradient for xylem
loading, is not very different from other types of root
cells. Root stelar cells do have a plasma membrane proton
pump to assist in generating and maintaining a membrane
potential (Clarkson, 1993, and references therein).
There are some reports of the toxic effects of nitrate
Nitrate transport and compartmentation in cereal roots
and this may be the reason for controlling cytosolic
concentration. At high concentrations the ion has nonspecific chaotropic effects (Griffith et al., 1986; Weiser
and Bentrup, 1994), while even at concentrations below
10 mol m~\ nitrate specifically inhibits proton pumping
by the vacuolar ATPase (Wang and Sze, 1985).
Mechanisms for regulating cytosolic nitrate
Cytosolic nitrate concentration in a root cell must be
determined by several processes, including transport at
both the plasma membrane and tonoplast, assimilation
and symplastic transport to the xylem parenchyma for
transport to the shoot.
Cytosolic nitrate concentration must be maintained by
the steady-state between processes at both the plasma
membrane and the tonoplast. At the plasma membrane,
the steady-state between influx and efflux will not only
determine net uptake, but also influence cytosolic nitrate
concentration. Indeed this might provide an explanation
for the energetically wasteful process of nitrate efflux. It
has long puzzled transport physiologists why the plant
cell invests energy in the process of nitrate influx only to
allow the ion to efflux from the cell. Nitrate efflux may
be important for maintaining cytosolic nitrate. At the
tonoplast of nitrate replete cells, active transport is needed
to account for the concentration of nitrate inside the
vacuole, while an open channel will allow nitrate ions to
move passively into the cytosol. Overall regulation of
cytosolic nitrate requires co-ordination of all of these
processes at both membranes and the most direct way of
achieving this is to make the nitrate transport processes
sensitive to cytosolic nitrate concentration. In the long
term (hours) the number and activity of the nitrate
cotransporters can be modified. Whereas, short-term control (minutes) can best be achieved by involving the
processes which can respond rapidly, that is passive efflux
(channels) at the plasma membrane and transport out of
the vacuole. In other words control occurs at the 'leak'
rather than the 'pump'; this view for control of net nitrate
uptake was advanced by Deane-Drummond (1984). Such
regulation would require direct interaction between cytosolic nitrate status and channel activity, perhaps by
phosphorylation of the channel like the a-TIP aquaporin
(Maurel et al., 1995). Anion channel blockers could be
used to investigate the role of the channels in maintaining
cytosolic nitrate concentration.
Experiments using lines of barley deficient in NR
structural genes have shown that NR has no role in
induction or in determining the kinetics of net nitrate
uptake (Warner and Huffaker, 1989). The reported values
for the Km of NR for nitrate are 0.12-0.6 mol m" 3
(KJeinhofs et al., 1989) which is much lower than the
cytosolic levels of nitrate achieved by active transport
(Table 2). This suggests that nitrate assimilation is not
851
determining the levels of cytosolic nitrate even though in
many species NR activity increases with nitrate supply
(Aslam et al., 1993; Fedorova et al., 1994). However,
under anaerobic conditions NR activity increases and
may then influence cytosolic nitrate levels, and this is the
basis for one of the methods for measuring cytosolic
nitrate and under these conditions nitrate supply determines NR activity (King et al., 1992). Measurements of
cytosolic nitrate in these NR-deficient mutants will establish if assimilation has any role in regulating cytosolic
nitrate activity.
Xylem loading may be involved in the regulation of
cytosolic nitrate concentration because excised roots show
a decrease in cytosolic nitrate (Zhen et al., 1992), but
excised roots can continue to produce xylem exudate
which contains nitrate (Behl etal., 1988). It seems unlikely
that the only mechanism maintaining cytosolic nitrate
concentration should be dependent on transpiration
because water supply is so variable. Measurements of
cytosolic nitrate in cultured single cells offer a simplified
system and should establish if xylem loading has any part
in regulation.
One intriguing question resulting from the regulation
of cytosolic nitrate, is how changes in external supply can
alter vacuolar nitrate accumulation? One possibility is
that the external nitrate concentration is somehow sensed
by proteins in the plasma membrane or the cell wall and
that messages are relayed to the nucleus to affect changes
in transport at the tonoplast. A nitrate sensor to initiate
the induction of uptake has also been proposed
(Redinbaugh and Campbell, 1991) and this may be the
role of reported plasma membrane associated NR activity
(Stdhr et al., 1995). An alternative explanation, not
requiring an environmental nitrate sensor, is that the
steady-state of the tonoplast transport system (efflux and
influx) adjusts to maintain cytosolic nitrate concentration
very efficiently, so that as more nitrate enters the cytosol
then more accumulates inside the vacuole. This model
also requires that the tonoplast nitrate transport system
is able to sense and respond to very small changes in
cytosolic nitrate activity which have not been detected by
microelectrode measurements. In a mature cell, the cytosol is a small volume spread thinly around the vacuole;
an arrangement which will favour tonoplast transport as
an important mechanism for the regulation of cytosolic
nitrate.
Conclusions and future work
In conclusion, nitrate-selective microelectrodes presently
offer the most reliable method for measuring cytosolic
and vacuolar nitrate concentration. The method also
provides useful data on the thermodynamic gradients of
nitrate. The measurements show that nitrate uptake across
the plasma membrane, even at the relatively high external
852 Miller and Smith
concentration of 10 mol m~3, must be by an active
process in epidermal cells of barley and maize roots.
Nitrate uptake at the plasma membrane could utilize the
transmembrane pH gradient by symport of protons and
nitrate; only at an external pH > 7.5 might such a mechanism be unable to maintain cytosolic nitrate concentration.
More ion-selective microelectrode measurements of the
electrochemical gradients at such pH extremes are needed
to investigate the feasiblity and limitations of this transport mechanism. It remains to be elucidated if cytosolic
nitrate activity is the signal for the induction of nitrate
transport and assimilation genes as all microelectrode
measurements have been made on nitrate-induced barley
root cells. Triple-barrelled nitrate-selective microelectrodes could be used to measure the changes in cytosolic
nitrate during the process of induction.
A combination of molecular and biophysical techniques
can be used to tease apart the processes involved in the
regulation of cytosolic nitrate. As more transporter genes
are identified it becomes feasible to manipulate their
expression in transgenic plants with the aim of studying
nutrient ion transport and compartmentation. However,
as the cytosolic nutrient ion concentrations are regulated
the most likely outcome of such manipulation is that the
cell will adjust other transport systems to compensate
and so maintain cytosolic homeostasis.
Acknowledgements
We wish to thank Wendy Gregory for her help with tissue
analysis of nitrate and Karen Moore for assistance with
statistical analysis and Roger Leigh for critically reading the
manucript. 1ACR receives grant-aided support from the
Biotechnology and Biological Sciences Research Council of the
United Kingdom.
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