Planta
Planta (1991) 185:356-361
9 Springer-Verlag1991
Compartmental nitrate concentrations in barley root cells measured
with nitrate-selective microelectrodes and by single-cell sap sampling
Rui-Guang Zhen 1, Hans-Werner Koyro 2, Roger A. Leigh 1, A. Deri Tomos 2, and Anthony J. Miller 1.
1 Biochemistry and Physiology Department, AFRC Institute of Arable Crops Research, Rothamsted Experimental Station, Harpenden,
Herts AL5 2JQ, UK, and
2 School of Biological Sciences, University College of North Wales, Bangor, Gwynedd LL57 2UW, UK
Received 23 May; accepted 4 July 1991
Abstract. Nitrate-selective microelectrodes were used to
measure intracellular nitrate concentrations (as activities) in epidermal and cortical cells of roots of 5-d-old
barley (Hordeum vulgare L.) seedlings grown in nutrient
solution containing 10 mol 9m-3 nitrate. Measurements
in each cell type grouped into two populations with mean
(•
values of 5.4+0.5 m o l - m -3 (n=19) and
4 1 . 8 + 2 . 6 m o l . m -3 ( n = 3 5 ) in epidermal cells, and
3.2•
-3 ( n = 4 ) and 7 2 . 8 •
-3
(n = 13) in cortical cells. These could represent the cytoplasmic and vacuolar nitrate concentrations, respectively, in each cell type. To test this hypothesis, a single-cell
sampling procedure was used to withdraw a vacuolar sap
sample from individual epidermal and cortical cells.
Measurement of the nitrate concentration in these samples by a fluorometric nitrate-reductase assay confirmed a mean vacuolar nitrate concentration of 52.6 •
5.3 tool" m -3 (n= 10) in epidermal cells and 101.2•
4.8 mol 9m-3 (n = 44) in cortical cells. The nitrate-reductase assay gave only a single population of measurements
in each cell type, supporting the hypothesis that the
higher of the two populations o f electrode measurements
in each cell type are vacuolar in origin. Differences in the
absolute values obtained by these methods are probably
related to the fact that the nitrate electrodes were calibrated against nitrate activity but the enzymic assay
against concentration. Furthermore, a 28-h time course
for the accumulation o f nitrate measured with electrodes
in epidermal cells showed the apparent cytoplasmic
measurements remained constant at 5.0 • 0.7 mol 9 m-3,
while the vacuole accumulated nitrate to 30-50 tool 9m - 3.
The implications of the data for mechanisms of nitrate
transport at the plasma membrane and tonoplast are
discussed.
Key words: Hordeum (intracellular nitrate)
Nitrate
compartmentation (cytosol, vacuole) - Root (intracellular nitrate)
* To whom correspondence should be addressed
Symbol." X~= Chi-squared with n degrees of freedom
Introduction
Nitrate is the chief storage form o f nitrogen in the vacuole and can accumulate in some tissues to concentrations as high as 300 mol - m -3 (Hewitt et al. 1979). The
accumulation of nitrate has important physiological implications because stored nitrate can serve as an important osmoticum for turgor generation and also as a
storage pool to meet the needs of plants during conditions of nitrogen shortage. Importantly in field grown
crops, the accumulation of nitrate within the plant vacuole can offer a route to remove the available nitrate
from the soil and thus help to prevent nitrate leaching
into groundwater (Addiscott and Darby 1991). Large
accumulations of nitrate in the vacuole could imply an
active mechanism of transport across the tonoplast into
the vacuole, but before attempting to describe the possible mechanism of transport at either the tonoplast or the
plasma membrane it is necessary to measure intracellular
compartmental concentrations o f nitrate. Siddiqi et al.
(1990) suggested that uptake at high external concentrations o f nitrate could be by a passive mechanism, proposing a nitrate channel at the plasma membrane; however,
the energetics of such a mechanism would require micromolar cytoplasmic nitrate concentrations.
For higher plants, vacuolar nitrate concentration can
be estimated by chemical analysis of fully vacuolated
tissues because the vacuole dominates such measurements (Jeschke and Stelter 1976; Hajibagheri et al. 1988).
However, cytoplasmic nitrate concentration is more difficult to obtain. Estimates for barley roots have been
made using the technique of compartmental tracer-flux
analysis with 13N-labelled nitrate (Lee and Clarkson
1986) or with the nitrate analogue 36CIO3 (DeaneD r u m m o n d and Glass 1982). These two different tracers
have yielded cytoplasmic nitrate concentrations of 26
and 8 mol" m-3, respectively. It is not known whether
these differences are due to errors in the tracer-flux method or to experimental treatments imposed on the tissues.
Other approaches to estimating cytoplasmic nitrate concentration in barley include tissue-fractionation proce-
R.-G. Zhen et al.: Nitrate concentrations in barley-root cell compartments
dures ( M a r t i n o i a et al. 1986). These yielded c y t o p l a s m i c
n i t r a t e c o n c e n t r a t i o n s o f a r o u n d 4 m o l - m -3 for leaf
m e s o p h y l l cells.
I n this p a p e r we report direct m e a s u r e m e n t s o f intracellular n i t r a t e c o n c e n t r a t i o n s in barley r o o t cells using nitrate-selective microelectrodes. W e recently reported the d e v e l o p m e n t o f these electrodes a n d their use to
m e a s u r e n i t r a t e in c y t o p l a s m a n d vacuole o f giant algal
cells (Miller a n d Z h e n 1991). F o r these g i a n t algal cells,
the microelectrode m e a s u r e m e n t s could be checked b y
chemical analysis o f the whole cells. This was o b v i o u s l y
n o t possible for i n d i v i d u a l r o o t cells so we checked the
microelectrode results by u s i n g the single-cell s a m p l i n g
t e c h n i q u e o f M a l o n e et al. (1989, 1991) to r e m o v e vacu o l a r sap from cells a n d t h e n m e a s u r e d n i t r a t e in these
samples u s i n g a n e n z y m i c procedure.
Material and methods
Barley (Hordeum vulgare L. cv. Klaxon) seeds were germinated and
grown for 3-4 d on filter paper moistened with 0.2 m o l - m -3
CaSO4 and then transferred to a modified Hoagland's nutrient
solution containing 2 m o l ' m - 3 Ca(NO3)2 ' 1 . 2 m o l ' m - 3
KH2PO4, 6 mol - m -3 KNO3, 2.4 mol - m -3 MgSO4, 5 mol - m -3
CaCI2, 0.29 mol - m - 3 FeNa 9EDTA plus micronutrients as given
by Hoagland and Arnon (1950). The final nitrate concentration of
10mol' m -3 represents the upper limit for nitrate in the crop
environment (Barraclough 1986) and was chosen to achieve a high
vacuolar accumulation of nitrate. Seedlings were grown in aerated
water culture at 20~
under a photon fluence rate of
450-480 gmol - m - 2. s- 1, three seedlings each in 1 dm 3 of culture
medium with the roots maintained in darkness.
Double-barrelled nitrate-selective
microelectrodes
were
prepared by the method of Miller and Zhen (1991) except that tip
diameters were less than 1 gm. Both barrels of the electrodes had
resistances of 10 to 20 Mf~ when filled with 100 mol" m -3 KC1
solution. An hour before use, both barrels were backfilled;
100mol-m -3 KC1 in the membrane-potential-measuring barrel
and 100 tool 9m- 3 NAN03 + 100 mol 9m - 3 KCI in the nitratesensing barrel. Intracellular nitrate measurements were made using
a high-input impedance differential electrometer (model FD 223;
World Precision Instruments, New Haven, Conn., USA). The electrometer output passed via an A/D converter (Labmaster DMA/
PGH; Scientific Solutions, Solon, Ohio, USA) at a sampling frequency of 10 Hz to an Opus PC V microcomputer. Data were
analysed using the VISER software developed by I.R. Jennings,
Biology Department, University of York, UK.
Before making intracellular measurements the nitrate-selective
microelectrodes were calibrated in nitrate solutions containing
varying activities of K2HPO 4 to give a constant background ionic
strength, and buffered with 4-(2-hydroxyethyl)-l-piperazineethanesulphonic acid (Hepes), pH 7.9 (Miller and Zhen 1991). Nitrate
activities were calculated using the SOLCON programme (written
by D.C.S. White, Biology Department, University of York, UK).
During microelectrode measurements the intact roots were perfused
with the nutrient solution containing 10 mol. m -3 nitrate. All
impalements were made on ceils 1 to 2 cm from the root tip and
measurements were only accepted if the ceils had a membrane
potential more negative than - 4 0 mV (85% of impalements) and
if the microelectrodes recalibrated after insertion into the cell (e.g.
inset to Fig.l). For cells included in this study, the membrane
potentials ranged between - 4 0 and - 128 mV with a mean + SE of
-- 71 + 1.9 mV (147 measurements). These values are comparable to
membrane potentials measured in root cells in other studies (e.g.
Pitman et al. 1971; Bowling 1972; Mertz and Higinbotham 1976).
Samples of vacuolar sap of 10-25 pl were withdrawn from
individual cells using a modified pressure probe and were kept
357
under water-saturated silicone oil at 4~ (Malone et al. 1989).
Nitrate was measured in the samples using an NADPH-linked
nitrate-reductase assay (Von Beutler et al. 1986) conducted on a
microscope slide at room temperature using a microscope fluorometer (Leitz MPV Compact 2 Microscope Photometer and MPV
software (Leitz, Wetzlar, FRG) with a Vig 1 computer (Viglen,
London, UK)). In this assay, nitrate is reduced by NADPH-nitrate
reductase to nitrite and the amount of NADPH oxidised during the
reaction is dependent on the concentration of nitrate. The decrease
in NADPH is measured by the change in fluorescence (using Leitz
filter block A, excitation at 270-380nm and emission at
410-580 rim). The assay involved placing 50-pl droplets of assay
buffer solution, containing 91 mol" m -3 imidazole-HCl(pH 7.8),
10.5 mmol 9m -3 FAD-Na z and 4.1 mol - m -3 NADPH, under
water-saturated 2:1 (v/v) paraffin/hexadecane oil in a 1- to 2-ramdeep aluminium ring on a glass microscope slide using a glass
constriction pipette. A sample of vacuolar sap (10-20 pl) or a
standard amount of nitrate was then added to the droplet using a
constriction pipette and the fluorescence (Fo) was measured, Finally, 30 pl of nitrate reductase (4.5 U" ml-1 from Aspergillus,
Boehringer-Mannheim, Mannheim, F.R.G.) was added to each
droplet. After 40 rain. when the reaction had reached an endpoint,
the final fluorescence (F1) was measured. The change in fluorescence
(AF = F 1 - Fo) was proportional to the nitrate concentration initially present in the samples over the range 5 to 50 tool 9m 3. All
samples were brought into the linear range by dilution. At all times
the same volume was added to the assay droplet.
Total nitrate was extracted from roots with boiling water and
was measured by ion chromatography (column AS4A with an AG4
guard column; Dionex (UK) Camberley, Surrey, UK). The water
content of roots was calculated from the difference between fresh
and dry weights. Fresh weight was determined after the roots had
been rinsed briefly in deionised water to remove nutrient solution
and then gently blotted with tissue paper. Roots were dried at 80~ C
for 12 h. All chemicals were analytical grade and were from Fluka
Chemicals, Glossop, Derbyshire, UK.
Results
F i g u r e 1 shows a typical r e c o r d i n g f r o m a b a r l e y - r o o t
e p i d e r m a l cell i m p a l e d with a d o u b l e - b a r r e l l e d nitrateselective microelectrode. T h e i n t r a c e l l u l a r n i t r a t e activity
was calculated f r o m the line fitted t h r o u g h the c o m b i n e d
" b e f o r e " a n d "after" c a l i b r a t i o n d a t a p o i n t s (inset,
Fig. 1) u s i n g a simplified N i c o l s k y - E i s e n m a n e q u a t i o n
(Miller a n d S a n d e r s 1987).
N i t r a t e activities m e a s u r e d in 54 e p i d e r m a l cells f r o m
roots g r o w n in 1 0 t o o l . m -3 n i t r a t e for 2 4 - 3 0 h fell
w i t h i n the r a n g e 2 to 85 tool 9m - 3 a n d clustered i n t o two
p o p u l a t i o n s (Fig. 2). This was c o n f i r m e d b y statistical
analysis which showed t h a t all the d a t a p o i n t s did n o t fit
a single n o r m a l d i s t r i b u t i o n (Z92= 52.65) b u t c o u l d be
described b y two n o r m a l l y d i s t r i b u t e d p o p u l a t i o n s
(Z~ = 4.12). O n e g r o u p (designated c o m p a r t m e n t 1) h a d
a m e a n value ( 9 SE) o f 5.4 + 0.5 tool 9m - 3 a n d the other
( c o m p a r t m e n t 2) 4 1 . 8 •
t o o l . m -3 (Table 1). T h e
m e a n m e m b r a n e p o t e n t i a l s ( ~ SE) of these two p o p u l a tions were - 7 3 _ + 6 m V for the lower n i t r a t e activities
a n d - 65_+ 4 m V for the higher.
I n a n o t h e r experiment, m e a s u r e m e n t s were m a d e o n
r o o t e p i d e r m a l cells at v a r i o u s times after the transfer o f
roots to a s o l u t i o n c o n t a i n i n g 10 t o o l . m -3 n i t r a t e (see
Fig. 3). Low n i t r a t e activities ( < 8 tool 9m - 3 ) were measured in cells o f the roots which h a d b e e n in n i t r a t e
s o l u t i o n for less t h a n 6 h, b u t thereafter m e a s u r e m e n t s
fell into two groups. O n e set o f m e a s u r e m e n t s gave ac-
358
R.-G. Zhen et al.: Nitrate concentrations in barley-root cell compartments
140mV
1
~a
=,->
E~E
o~-~,
t
2
3
4
- ~ [NOi]
5
6
o "-" -50J
.2
0.
-100
Z~
Z
0
2o!
.,.
OJ
1 rain
Fig. I. Recordings for a barley-root epidermal cell impaled with a
double-barrelled nitrate-selective microelectrode. The cell was impaled about 30 s after the start of recording. The upper trace shows
output from the membrane-potential-sensing barrel (mV), the
middle, output from the nitrate-sensing barrel (mV) and the
lower, nitrate concentration (mol 9m-3) calculated by the VISER
programme. The nitrate activity in this cell compartment was
4.0 tool 9m 3. The inset shows the calibration data before (o) and
.o
14
>
12
e~
10
8
6
4
E
z
2
0
ffl
.Q
o
0
I
2
4
// J-- - 1
8
10 10
6
after (e) impalement. The curve was fitted to the combined data
with a simplified Nicolsky-Eisenman equation of the form
E = Eo - S log {[NO;-] + K}, in which E is the measured output of
the nitrate-selective electrode, Eo is the constant electrode reference
potential, S is the slope and K is a term which subsumes the
concentrations of the interfering ions and the selectivity coefficients
of the electrode for those ions. Fitted parameter values were
E0 = - 7 5 . 7 mV; S = 53.8 mV; K = 196 mmol 9m -3
Fig. 2. Histogram showing the distribution of
nitrate concentrations measured with the nitrate-selective microelectrode in epidermal
cells of barley roots grown for 24 to 30 h in
nutrient solution containing 10 mol - m -3 nitrate. A total of 54 measurements were made,
each on different cells from 33 different plants
over a two-month period. Mean values_+ SE
for the two populations are 5.4+0.5 and
41.8_+2.6 mol 9m -3
20
30
40
50
80
N i t r a t e (mol.m -3)
Table 1. Intracellular nitrate concentrations in barley root cells
measured using nitrate-selective microelectrodes. All roots were
grown for 24 to 30 h in nutrient solution containing 10 mol 9m -3
nitrate, and measurements were made 1 2 cm from the root tip. The
epidermal cells were identified as the first layer of cells encountered
by the micropipette; cortical cells were sampled from the two layers
of cells beneath the epidermis. All values are given as mean_+ SE
Cell type
Cell
compartment
Number of
measurements
Nitrate
activity
(mol. m - 3)
Membrane
potential
(mV)
Epidermal
1
2
19
35
Cortical
l
2
4
13
5.4•
41.8•
3.2•
72.8•
--73•
--65•
--86•
--71•
tivities w h i c h r e m a i n e d r e l a t i v e l y c o n s t a n t a n d w e r e less
t h a n 10 m o l 9 m - 3 n i t r a t e , b u t t h e o t h e r i n c r e a s e d w i t h
t i m e a n d a f t e r 16 h m o s t v a l u e s fell i n t o t h e r a n g e
30 50 m o l 9 m - 3 (Fig. 3). T h i s p o p u l a t i o n o f v a l u e s inc r e a s e d at a r a t e o f 1 . 6 m o l - m - 3 - h -1, o b t a i n e d b y
fitting a l i n e a r r e g r e s s i o n line (line n o t s h o w n ) f o r d a t a
6
4
7
f r o m 7.5 to 26 h a f t e r t r a n s f e r o f t h e r o o t s to n i t r a t e .
C h a n g i n g the l i g h t r e g i m e f r o m a 16-h d a y l e n g t h to
c o n t i n u o u s light h a d n o effect o n this d i s t r i b u t i o n o f
n i t r a t e a c t i v i t i e s (Fig. 3).
F o r g i a n t a l g a l cells, c h e m i c a l a n a l y s i s o f t h e w h o l e
cells g a v e a m e a s u r e o f v a c u o l a r n i t r a t e c o n c e n t r a t i o n
R.-G. Zhen et al. : Nitrate concentrations in barley-root cell compartments
6O
Table 3. Concentrations of nitrate in vacuolar sap isolated from
E
epidermal and cortical cells of barley roots using a single-cell sampling procedure. The roots had been grown in 10 mol 9m -3 nitrate
for 24 to 30 h and cells were located 1-2 cm from the root tip.
Nitrate was measured using nitrate reductase as described in
Material and methods. All values are given as mean + SE
0
-~ 5 0
E
0
0
g 40
0
".~
9
0
oo 9
9
0
c
0
0
359
Cell type
Number
sampled
Nitrate
concentration
(mol" m- 3)
Epidermal
Cortical
10
44
52.64- 5.3
101.24- 4.8
0
0
20
10
o~
9 9 o
qp 9
Z
I
0
4
8
12
I
I
16
9
I 01
9I
20
I
24
I
28
Time in nitrate (h)
Fig. 3. The time course of nitrate accumulation in barley-root
epidermal cells measured using nitrate-selective microelectrodes.
Plants were grown in 10mol-m -3 nitrate under either a 16-h
daylength (9 or in continuous light (o)
Table 2. Nitrate concentrations in whole barley roots and at varying
distances from the root tip. The roots were grown in 10 mol' m -3
nitrate for 26 to 28 h under continuous light and then nitrate was
extracted and determined by ion chromatography. Concentrations
were calculated relative to the water content of the roots. Each
analysis is the mean • SE of three samples
Distance from root tip
(cm)
Nitrate concentration
(mol. m -3)
0-2.5
2.5-5.0
>5.0
Wholetissue
90.9•
113.9•
93.3•
100.1•
(Miller and Zhen 1991), so a similar approach was used
initially for barley roots. Table 2 shows the results of
chemical analysis o f roots from barley plants grown in
10 mol 9m -3 nitrate for 26 to 28 h in continuous light.
The average nitrate concentration in the whole roots was
100 mol 9m-3, much higher than either of the two electrode populations in Fig. 2. Nevertheless, as there are
gradients of nutrients accumulated along the root in
some species (Belton et al. 1985) and all our electrode
measurements were made 1-2 cm from the root tip it was
possible that the discrepancy arose because cells more
distal from the root tip had higher nitrate concentrations
and dominated the whole-root measurements. Therefore,
a more detailed analysis was made of nitrate concentration along the roots. Table 2 shows there was some
evidence of a nitrate gradient along the barley roots, with
the highest nitrate concentrations in the region 2.5 to
5 cm from the tip, but nitrate levels in all segments were
still much greater than either of the two groups of measurements obtained using the nitrate-selective microelectrodes. This indicated that there could be a nitrate gradient across the root, possibly between the epidermal and
cortical cells.
This gradient was confirmed when nitrate activities in
cortical cells were measured. As in the epidermal cells,
two populations of nitrate activities were found
(Table 1). The lower one (compartment 1) had a mean
value o f 3.2 mol 9m - 3 and was not significantly different
from the value of the lower population in the epidermal
cells. However, the higher population (compartment 2)
had a mean value of 72.8 mol 9m - 3 , significantly greater
than that of the corresponding population in the epidermal cells. However, these cortical nitrate activities were
still insufficient to explain completely the whole-root
nitrate concentrations. Nonetheless, if it is assumed that
the whole-root nitrate concentrations are generally dominated by the vacuolar nitrate concentrations, then these
results are consistent with compartment 2 being the vacuole.
Further evidence that compartment 2 might be the
vacuole was obtained using a single-cell sampling
procedure to sample vacuolar sap from individual epidermal and cortical cells and then assaying nitrate using
a nitrate-reductase-based fluorescence assay. The nitrate
concentrations in the single-cell sap samples were normally distributed into single populations for each cell
type and the mean values are shown in Table 3. The
concentrations in the epidermal cells were about 13%
higher than the mean value for compartment 2 measured
with nitrate-selective microelectrodes (compare Tables 1
and 3). Those in the cortical cells were about 40% higher.
Nonetheless, it is clear that the sap samples, which consist mainly of vacuolar sap (Malone et al. 1991) never had
nitrate concentrations corresponding to those in compartment 1 in either epidermal or cortical cells.
Discussion
The simplest explanation of the observation that both
epidermal and cortical cells contain two populations of
nitrate activities is that these represent cytoplasmic and
vacuolar nitrate activities. The possibility that these populations derive from two distinct types o f epidermal or
cortical cells which differ in their ability to accumulate
nitrate is discounted by the results with the single-cell
sampling procedure which never gave values in the same
range as the lower population o f measurements in either
cell type. It would be unlikely that in a total of 54 cells
sampled, none would have come from cells with the
lower nitrate concentrations, if these existed. The higher
nitrate concentrations measured in the isolated sap sam-
360
R.-G. Zhen et al.: Nitrate concentrations in barley-root cell compartments
ples are p r o b a b l y because these sap samples were calibrated using nitrate concentrations whereas the nitrateselective microelectrodes are calibrated using nitrate activities. The difference between the results obtained for
each method could be explained if the activity coefficient
in vivo for nitrate was a b o u t 0.75. The activity coefficient
of 100 mol - m -3 K N O 3 is 0.74 (Weast 1974).
The intercellular gradient of vacuolar nitrate across
the root tissue m a y arise because most of the nitrate
reductase is present in the outer layer of cells (Rufty et
al. 1986). However, one limitation of measurements of
nitrate made in cortical cells either with microelectrodes
or by single-cell sampling is that the epidermal cells must
be damaged in order to reach these cells. The resulting
damage to the epidermis could change the distribution of
ions within cortical cells. Nevertheless, the electrode
measurements showed stable nitrate activities within cortical cells, indicating there were no significant changes in
nitrate distribution while measurements were made.
I f the lower populations of measurements made with
the nitrate-selective microelectrodes are cytoplasmic in
origin, as seems likely, then they indicate that nitrate
concentrations in this c o m p a r t m e n t are in the millimolar
range and are consistent with values obtained by compartmental tracer flux analysis. After the roots had been
in a nutrient solution containing 10 m o l - -3 nitrate for
14 h or more the cytoplasmic nitrate activity in both
epidermal and cortical cells had reached a steady-state
value o f a b o u t 5 mol 9 m - 3 (Table 1, Figs 2 and 3). This
cytoplasmic nitrate activity must represent a steady state
between nitrate assimilation, partitioning to the vacuole
and nitrate m o v e m e n t via the root symplast to the shoot.
The cytoplasmic nitrate reductase has a K~ for nitrate of
0.12 to 0.6 mol 9m -3 (Kleinhofs et al. 1989) so cytoplasmic nitrate concentration is not limiting nitrate assimilation in these root cells at an external nitrate concentration o f 10 tool 9m -3.
The results in Fig. 3 support the view that there is
constitutive nitrate uptake at the plasma m e m b r a n e in
non-induced barley roots because after only a few hours
a millimolar concentration of nitrate in the cell has been
reached. The accumulation of nitrate in these cells arises
without a lag, which would be required for the de-novo
synthesis o f new p l a s m a - m e m b r a n e nitrate-transporter
proteins. The nature of this nitrate transporter remains
unclear, but the observation that cytoplasmic nitrate
concentrations are probably in the millimolar range does
not support the passive model proposed by Siddiqi et al.
(1990) for uptake at the plasma m e m b r a n e at high external nitrate concentrations. This requires cytoplasmic nitrate concentrations in the micromolar range. It would
seem that nitrate transport at the plasma m e m b r a n e must
be an active process. An active mechanism of nitrate
accumulation in the vacuole also becomes necessary after
8 h, if the cytoplasm fills to achieve an equilibrium concentration of 5 mol 9m - 3 and the trans-tonoplast potential is + 20 mV or less. A passive mechanism for nitrate
accumulation would require trans-tonoplast potentials
of a b o u t + 50 m V for epidermal cells and + 80 mV for
cortical cells whereas measured m e m b r a n e potentials
indicate that the trans-tonoplast potential is close to zero
(see Table 1). Several authors have reported that anion
transport at the tonoplast can occur via a passive mechanism dependent only on the trans-tonoplast potential
(Pope and Leigh 1990; Martinoia et al. 1986) but it
would appear that this cannot be the only mechanism of
nitrate accumulation in the vacuole in barley grown in
1 0 m o l . m -3 nitrate. Accurate and unequivocal measurements of the trans-tonoplast m e m b r a n e potential
will be required to confirm this.
The storage-root tonoplast H +-ATPase activity of the
red beet was shown to be inhibited by nitrate in vitro with
maximal inhibition occurring at 100 tool. m -3 (Walker
and Leigh 1981). Using the whole-cell patch-clamp
technique with isolated sugarbeet root vacuoles, Hedrich
et al. (1989) showed that this inhibition requires nitrate
to be present on the cytoplasmic side of the tonoplast. A
nitrate concentration of up to 200 mol 9m - 3 on the vacuolar side of the tonoplast was found not to inhibit the
H+-ATPase. Concentrations of nitrate between 1 and
10 m o l . m - 3 have no effect on the beet tonoplast H + ATPase (Walker and Leigh 1981) so at the concentration
of nitrate measured in the cytoplasm with the microelectrodes, the tonoplast H +-ATPase activity should not be
inhibited by nitrate.
In conclusion, the results in this paper confirm that the
nitrate-selective microelectrodes previously used to
measure nitrate concentrations in giant algal cells (Miller
and Zhen 1991) can be used successfully in higher-plant
cells. As in Chara corallina, the results indicate two populations of nitrate concentrations in barley root cells, and
these probably represent cytoplasmic and vacuolar concentrations, with the cytoplasmic concentration being
smaller and more constant than that in the vacuole. The
successful application of the electrodes to higher-plant
cells opens the possibilty of greater understanding of
nitrate dynamics and transport through direct measurements of nitrate concentrations in subcellular compartments in vivo.
R.-G.Z. was awarded a Sino-British Friendship Scholarship sponsored by the British Council and H.-W.K. was supported by an
AFRC Linked Research Grant to A.D.T for collaboration with
R.A.L. We wish to thank Dr. K. Goulding for advice on ion
chromatography, Dr. K. Moore for assistance with statistical analysis and Dr. J.H. Williams for advice on the microsample analysis.
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