Annals of Botany 57, 471-486, 1986 471

Effects of Nitrogen Deficiency on the Absorption

of Nitrate and Ammonium by Barley Plants
R. B. LEE 1 and KATHRYN A. RUDGE 2
Agricultural and Food Research Council Letcombe Laboratory, Wantage, Oxfordshire OX12 9JT, UK

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Accepted: 25 September 1985

ABSTRACT
When young barley plants which had been supplied with nitrate were deprived of this source of N, an enhanced
capacity for absorption of either nitrate or ammonium ions developed, reaching a maximum in about 3 d under
the particular experimental conditions used. The net uptake rate of either nutrient was then approximately
three times that in plants which had received nitrate throughout. Likewise, withholding external N from plants
previously growing with ammonium caused a 2-4-fold increase in their subsequent capacity to absorb that ion,
compared with control plants grown with an uninterrupted ammonium supply. Accelerated nitrate uptake in
N-starved plants was not accompanied by additional phosphate or sulphate absorption, but the plants had
the capacity to absorb more potassium, whether or not ammonium was also present in the solution. Indirect
evidence from analyses of root tissue suggests that these responses to mild N-stress may depend on some
property of an N fraction which does not include nitrate or ammonium.

Key words. Hordeum vulgare, barley, nitrogen, ammonium, nitrate, N-deficiency, absorption.

INTRODUCTION
Withholding a mineral nutrient from a growing plant for an appropriate period of time
often results in the plant developing an enhanced capacity to absorb that nutrient when
the supply is restored. This phenomenon has been studied in greatest detail with regard
to potassium, phosphorus and sulphur nutrition (see references in Clarkson and Hanson,
1980; Lee, 1982; Drew et al., 1984), although it is also shown with ions which have no
major nutrient function (chloride, bromide: Lee, 1982).
There are indications from the literature that nitrogen nutrition conforms to a similar
pattern, and examples of increased nitrate absorption following N-starvation, or of
decreased absorption following pre-treatment with nitrate, have been described in barley
(Humphries, 1951; Smith, 1973; Deane-Drummond, 1982), maize (Helder, 1952; Ivanko
and Ingversen, 1971; Mackown, Volk and Jackson, 1981), wheat (Jackson et al., 1976b;
Talouizte et al., 1984), ryegrass (Vose, 1962; Clement, Jones and Hopper, 1979), bean
(Breteler and Nissen, 1982), Arabidopsis (Doddema and Otten, 1979), tobacco cells
(Filner, 1969), Lemna and green algae (Ullrich, Schmitt and Arntz, 1981). Rather fewer
instances of accelerated ammonium uptake under these circumstances are known
(Chlorella: Syrett, 1953; wheat: Tromp, 1962, and Jackson, Kwik and Volk, 1976b;
Penicillium and Aspergillus: Hackette et al., 1970; maize: Ivanko and Ingversen, 1971;
Thalassiosira: McCarthy and Goldman, 1979). In the majority of these investigations,
there was little or no attempt to correlate quantitatively the rate of N-absorption with
the N-status of the plant material: parameters suitable for assessing the latter were
generally not measured.
1
To whom reprint requests should be sent. Present address: University of Bristol, Department of
Agricultural Sciences, Long Ashton Research Station, Bristol, BS18 9AF, UK.
2
Sandwich course student, University of Bath, UK.

0305-7364/86/040471 + 16 $03.00/0 © 1986 Annals of Botany Company

472 Lee and Rudge—Ammonium and Nitrate Absorption by Barley
Preliminary studies of nitrate absorption by intact barley plants (Lee, 1982) showed
that the response to N-deficiency was markedly different, in both magnitude and timing,
from the response to P- and S-deficiency. These differences have now been studied in
more detail; an outline of the results has already appeared (Lee and Rudge, 1983).

MATERIALS AND METHODS

Procedures for growing the plants were based closely on those previously described (Lee,
1982; Lee and Ratcliffe, 1983). Briefly, at the start of each experiment (day 0), barley
seed (Hordeum vulgare L., cv. Midas) was surface sterilized, washed and sown on moist

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filter paper. On day 2 the germinated seeds were transferred to stainless steel gauzes fitted
to black plastic tanks each containing 25 1 of aerated nutrient solution (see below). Each
gauze (30x30 cm) held approximately 100 seedlings. The tanks were placed in a
controlled enviroment cabinet at 20 °C, 70 per cent r.h., 440/tE m~2 s"1, 16 h
photoperiod.
The basal (N-free) nutrient solution contained (HIM) : potassium, 0-5; magnesium, 015;
calcium, 015; phosphate, 0-5; sulphate, 015; chloride, 0-3. Iron and minor nutrients
were included (Lee, 1982). Depending on the N-source required, either sodium nitrate
(1-5 HIM) or ammonium sulphate (0-75 HIM, to give 1-5 HIM ammonium ion) was added
to the basal solution. Powdered calcium carbonate was used to prevent acidification
during growth on ammonium-N: thefinalpH of the solution was 6-8. In one experiment
(Table 6 B), potassium-free nutrient was prepared by replacing potassium phosphate in
the basal solution with sodium phosphate at the same concentration.
When plants were to be starved of N, the nitrate- or ammonium-containing solutions
were replaced with fresh basal solution on the appropriate day. The nutrient solutions
for the control plants receiving N were also routinely renewed on day 8. Measurements
of ammonium or nitrate absorption were made on day 10.
Net uptake of ammonium or nitrate over 4-6 h (Tables 2, 3, 5, 6) was calculated from
the depletion of a solution containing 0-5 HIM potassium phosphate and 05 mM calcium
sulphate (pH 60), with either 0-2 mM potassium nitrate or 0-2 mM ammonium chloride
(except in Table 2 A, where the ammonium chloride concentration was 0-5 mM). Groups
of 6-8 intact plants were attached to plastic frames and their roots immersed in ~ 300 ml
of the appropriate uptake solution, which was aerated continuously. The experiments
were carried out in continuous light in the controlled environment cabinet where the
plants had been grown, beginning at least 12 h after the end of the last dark period. One
millilitre samples were removed from the uptake solution at 20 or 30 min intervals and
deep-frozen for later nitrate or ammonium analysis. Loss of solution by sampling, by
transpiration and by evaporation from the liquid surface was measured by weighing at
the beginning and end; losses due to transpiration and evaporation were assumed to
occur uniformly with time, and it was thus possible to estimate the volume of the uptake
solution immediately before each 1 ml sample was taken. From this and the analytical
results, the quantity of ammonium or nitrate remaining in the solution at each sampling
time could be obtained.
Details of other experimental procedures are given in footnotes to the Tables.
Techniques for extraction and analysis of nitrate and ammonium in plant material, and
in solutions, were as described by Belton, Lee and Ratcliffe (1985). The analytical method
for ammonium was insensitive to amide- or amino-N. Total N in dried plant material
was determined by an automated Dumas combustion method. Analysis of 15N
(Table 4) was by conventional mass spectrometry. For measurement of 32P and 35S
(Table 5), the dried plant material was dissolved in concentrated nitric acid with minimum
heating, and dried down on aluminium planchettes for counting in a low-background
/^-counter. 42K (Table 6) was counted in fresh plant material and solutions using a y-
spectrometer.
 Lee and Rudge—Ammonium and Nitrate Absorption by Barley 473

RESULTS
Under our experimental conditions, barley plants supplied with nitrate in the basal
nutrient solution maintained a constant relative growth rate (RGR, on a total dry weight
basis) of 0-26 d"1 between days 5 and 10 (roots only: 0-23 d"1). Withholding nitrate for
periods of up to 5 d allowed us to vary the nitrogen status of the plants fairly
reproducibly. Over the first 3 d of nitrate starvation (days 7-10), the concentration of
nitrate in both roots and shoots was approximately halved each day (Table 1), although
the RGRs scarcely changed (roots 0-27 d~\ shoots 0-28 d"1, measured between days 9 and
10). The ratio of root dry weight to shoot dry weight reached its maximum value (0-68)

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when the plants had been starved of nitrate for 3 d (cf. a root:shoot ratio of 0-53 in
control plants of the same age). Thereafter, the RGR of the N-starved plants decreased.

TABLE 1. Concentration of total N, nitrate and ammonium in barley plants during the
development of nitrogen deficiency
Total N concentration Nitrate concentration
(mg N g"11d. wt + s.e.) (jimol g-> f.i wt + s.e.)
rci IUU wiuiuuL Roots Shoots Roots Shoots
nitrate (d)
0* 46-6±10 (17) 54-8±0-9(17) 73-7 + 3-0(18) 63-4+1-9(18)
0-5 45-2 41-7
1 32-3, 33-3 44-2, 44-8 26-4 + 2-2(5) 20-9 + 1-9(5)
1-5 261 36-2 13 2 13-5
2 25-7 + 0-7(5) 37-3 + 0-9(5) 9-5 + 0-7(6) 11-6 + 0-9(6)
2-5 20-4 41-7 4-6 12-7
3t 19-3 + 0-6(12) 30-9 + 0-7(12) 3-5 + 0-7(14) 7-8+1-0(14)
4 17-7, 18-2 25-2, 26-6 0-2, 0-2 4-3, 6-5
5 16-7 250 0-2 3-5

The plants were all 10 d old when taken for analysis, and had been grown without nitrate for the last 0-5 d
(see Materials and Methods). The above data were pooled from analyses of 20 separate batches of plants, grown
under similar conditions. Where applicable, the number of analyses used to calculate a mean value is given
in brackets.
* Ammonium concentration (jimo\ g~' f. wt + s.e.): roots, 1-7 + 0-2 (6); shoots, 1-3 + 0-1 (6). t Ammonium
concentration (jimol g"1 f.wt±s.e.): roots, 0-8±0-05 (5); shoots, 0-9±0-l (5).

For the experiment in Table 2 A, the plants were grown with ammonium as N-source.
Their roots differed markedly from those grown on nitrate: the seminal axes were shorter
and there was a profuse development of laterals (cf. Becking, 1956). Dry-matter
accumulation in the roots was not exponential, the RGR decreasing from 0-22 d"1 on
days 6-7 to 0066 d"1 on days 8-9; whereas after transfer to an ammonium-free solution
root growth continued exponentially with RGR 0-22 d"1 (days 6-9). Decreased root
growth in the presence of ammonium was to some extent offset by increased shoot
growth, so that between days 9 and 10 the overall RGR (total dry weight basis) of plants
receiving ammonium was 0074 d"1, compared with OlOd"1 in plants starved of
ammonium for the preceding 3 d.
Net uptake of nitrate and ammonium was measured over periods of 4-6 h by following
the depletion of the external solution (see Materials and Methods, and Fig. 1 for
representative depletion curves). The timecourses of absorption of the two ions were
markedly different and the choice of data for purposes of comparison is therefore
somewhat arbitrary. As previously noted in cereals (Picciurro el al., 1967; Minotti,
Williams and Jackson, 1969 a; Jackson, Volk and Tucker, 1972) and also in other species
(Munn and Jackson, 1978; Breteleer, Hanisch ten Cate and Nissen, 1979), ammonium
474 Lee and Rudge—Ammonium and Nitrate Absorption by Barley

TABLE 2. Effect of nitrogen deficiency on ammonium absorption by barley: initial rates

and average rates over 3-4 h
Ammonium concentration
Initial total N in roots Rate of ammonium absorption
concentration in (/miol g"1 f. wt root) (/tmol h"1 g"1 f. wt root + s.e.)
Period without roots
N-supply (d) (mg N g~' d. wt) Initial Final Initial* Averagef

A. Plants grown with ammonium as N-source

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0 60-9 7-9 8-5 -5-8±0-4 5-8±O-7
1 47-3
2 40-3 2-3 28-7 20-5±3-6 11-5 + 0-5
3 33-2 1-9 270 21-8+1-6 ll-7±0-4
4 28-9 11 301 14-3 + 0-9 12-1+0-2
B. Plants grown with nitrate as N-source
0 490 n.d. 51 4-7±0-5 4-2±0-l
1 33-3 71 8-7 + 0-2 6-1+0-1
2 251 80 13-5±0-8 8-8±0-3
3 19-4 6-9 151+0-7 10-3 + 0-4
4 17-7 171 17-1+0-3 10-3 + 0-4

A. Ammonium absorption was measured from 0-5 HIM ammonium chloride in aerated potassium phosphate-
calcium sulphate buffer, pH 6 0 . Final pH of solutions: 5-75-6-4. Means are from three replicate groups of
plants, except in the 0 d treatment where there were two such replicates.
B. Ammonium absorption was measured from 0-2 mM ammonium chloride in buffer as above. Final pH
of solutions 5-45-6-2. Means are from four replicate groups of plants (n.d.: not determined).
* Rate measured over the first 0-5 h of ammonium absorption, f For ammonium-grown plants, the average
rate was measured over the first 4 h, during which the external ammonium concentration was > 0-32 mM. For
nitrate-grown plants, the average rate was measured while the ammonium concentration was 5 50 /iM,
typically over the first 3 h.

uptake began without detectable delay and the maximum rate was achieved during the
first 0-5 h (Fig. 1 A, C). In nitrate-grown plants, whether subsequently N-starved or not,
the rate of ammonium uptake remained almost constant from the beginning of the
experiment until the external ammonium concentration had fallen below 50 /iu, and
linear regressions of cumulative absorption on time accounted for 95-99 per cent of the
variance during this period (Fig. 1 A). Similarly, during ammonium uptake by plants
grown on ammonium and then starved of N, linear regressions accounted for 90-99 per
cent of the variance over a period of 4 h (Fig. 1C).
By contrast, the timecourse of nitrate uptake was strongly influenced by previous
N-nutrition (Fig. 1 B). In nitrate-grown plants starved of N for 3 d, there was a delay
of about 4 h before the maximum net rate of nitrate uptake was attained (Fig. 2 B),
consistent with the widely observed 'induction phase' of nitrate transport (see review
by Jackson, 1978). Plants grown on ammonium alone, and then starved of N for 2 d,
gave a broadly similar response when first supplied with nitrate (results not shown).
Control plants grown on 1-5 mM nitrate throughout achieved a steady rate of net nitrate
uptake about 1 h after transfer to the 200 fiM nitrate solution (Fig. 2 B). Here, induction
of nitrate transport is presumably not involved, and the delay might reflect a temporary
continuation of rapid nitrate efflux when the roots were transferred from 1-5 mM to 200 /IM
nitrate solution (Minotti el al, 1969a, b; Jackson et al., 1976*). There was evidence of
a similar, temporary efflux of ammonium on transferring plants from 1-5 mM to 500 /IM
ammonium solution (Table 2 A).
Nitrogen-deficient barley plants absorbed either ammonium or nitrate more rapidly
than did the corresponding N-sufficient controls. In plants previously grown on nitrate,
 Lee and Rudge—Ammonium and Nitrate Absorption by Barley 475

100
A B

= 0-75 3
S A * A
A
*• A
A A
0-50 o •
o * A

o • A
Z 0 •

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E o • A
#
o A
o A

0 4 0 4 0 4
Time (h)
FIG. 1. Net uptake of nitrate and ammonium by barley plants: depletion measurements. A, Depletion
of 0-2 mM ammonium solution by plants grown on nitrate throughout (#), or starved of nitrate for
4 d (O); B, depletion of 0-2 mM nitrate solution by plants grown on nitrate throughout (A), or starved
of nitrate for 2-5 d (A); C, depletion of 0-5 mM ammonium solution by plants grown on ammonium
throughout (#), or starved of ammonium for 3 d (O).

o 300
I

o 200
E

100

10 20 30 40 50 60
Total N in roots (mg N g~'d.wt)
FIG. 2. A, Absorption of ammonium (O) and nitrate (A) in relation to total N concentration in
roots of nitrate-grown barley plants. To allow for the delay in the induction of nitrate transport in
N-deficient plants, the values are based on the maximum rates of nitrate absorption as defined in
the footnotes to Tables 3 and 5, and on the average rates of ammonium uptake over the corresponding
period (thefirst3-4 h). The following, minimum rates of uptake were taken as the N-sufficient control
values: nitrate, 3-4/tmol h"1 g~' f. wt root; ammonium, 3-7 fimo\ h"1 g~' f. wt root; B, net rates of
uptake after 0-2 mM nitrate was supplied to barley plants grown on nitrate throughout (A), or starved
of nitrate for 3 d (A)- The bars indicate the average values of s.e. of mean.

22 BOT 57
476 Lee and Rudge—Ammonium and Nitrate Absorption by Barley
acceleration of ammonium uptake was greatest in plants which had been starved of
nitrate for 3-4 d, when the rate was 2-5—3-6 times that in the corresponding controls
(depending on whether the initial or average rate of uptake is considered - Table 2 B).
For nitrate, the combined results of several experiments suggested that the greatest
enhancement of absorption, up to 2-5 times the rate in the controls, occurred when the
plants had been grown without nitrate for 2-3 d (Table 3). N-starvation beyond 3 d did
not stimulate nitrate uptake further, and there was some indication that the rate fell
slightly.

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T A B L E 3. Rate of nitrate absorption by N-deficient barley plants: short-term measurements

Maximum rate of
Initial root nitrate
nitrate absorption*
Period without concentration Omol h"1 g"1
nitrate (d) (jimo\ g~' f. wt) f. wt root±s.e.)

0 106 3-4 + 0-1

75 3-7 + 0-1
67 5-6 + 0-3
63 4-6 + 0-1
1 28 6-1+0-1
19 6-3 + 0-2
1-5 13 6-8 + 0-1
2 10 7-8 + 0-2
9-6 8-5 + 0-lf
9-3 7-5 + 0-1
2-5 4-6 7-7±0-l
3 2-2 7-9 + 0-1$
1-4 7-3 + 0-1
4 0-2 7-5 + 0-1
5 0-2 7-0 + 0-1

Nitrate absorption was measured from 0-2 miu potassium nitrate in aerated potassium phosphate-calcium
sulphate buffer, pH 6-0. Final pH of solutions: 6-2-6-4. Means are from four tofivegroups of replicate plants.
* Rate measured over the 2 h period where gradient of the depletion curve was steepest while the external
nitrate concentration remained ^ 50 (M, typically 2-7-4-7 h from the start. | Initial total N concentration
in root: 24-7 mg N g"1 d. wt. f Initial total N concentration in root: 19-2 mg N g"1 d. wt.

The situation in plants grown with ammonium as N-source was less clear-cut because
of possible incipient toxicity in the controls. However, ammonium uptake seemed to
reach a broad maximum after 2-4 d of N-starvation (Table 2 A), and in these plants the
enhancement of uptake capacity, whether expressed on a root dry weight basis or on
a 'per plant' basis (uptake 2-4 times that in controls), considerably exceeded the
improvement in RGR compared with the control plants still receiving ammonium (RGR
on total dry weight basis: 010 and 0074 d"1 in N-starved and control plants,
respectively, over days 9-10).
To investigate the persistence of the effect, we measured the net uptake of 15N-nitrate
or 15N-ammonium over 24-25 h (Table 4). Again, the rate of uptake of either nutrient
was increased in plants with lowered initial N-status, the maximum rates being reached
after 2-3 d of nitrate starvation. Thereafter, as N-stress became more severe, the rate
of net nitrate uptake showed signs of decreasing, and in two further experiments on plants
deprived of nitrate for 5 d the rate was 15 percent lower than in the N-sufficient controls;
while after 10 d without nitrate the rate was 35 per cent lower, and the roots contained
only 9-4 mg total N g~l dry weight.
 Lee and Rudge—Ammonium and Nitrate Absorption by Barley 411

TABLE 4. Absorption of anmonium and nitrate by N-deficient barley plants:

measurements over 24-25 h

Nitrate absorbed Ammonium absorbed

during uptake during uptake
Initial root period (jimo\ g~l period (/ttnol g~'
Period without concentration final f. wt final f. wt
nitrate (d) (jimoX g"1 f. wt) root + s.e.) root + s.e.)

Experiment A

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0 75 139 + 5 154 + 5
1 24 205 ±4 207 + 6
2 11 228 + 8 217 + 5
3 10 241+6 241+2

Experiment B
0 64 158 + 5
0-5 45 175 + 9
1 29 211+9
2 10 242 + 7
3 4 201 ± 8

Experiment A: Absorption of ammonium was measured over 25 h from aerated potassium phosphate-calcium
sulphate buffer, pH 6 0 , containing either 1-5 HIM [15N]ammonium chloride (30 atom per cent) or 1-5 mM
potassium 16 N-nitrate (30 atom per cent). Means are from five replicates, each consisting of two plants. Initial
concentration of ammonium in roots (/*mol g~l f. wt): 0 d, 2-6; 1 d, 0-9; 2 d, 1-2; 3 d, 0-7.
Experiment B: Nitrate absorption was measured over 24 h from aerated solution containing 0-5 mM
potassium phosphate and 0-75 mM calcium [16N]nitrate (1-5 mM nitrate, 10 atom per cent), pH 60. Means are
from eight replicate plants.

TABLE 5. Effect of nitrogen deficiency on simultaneous absorption of nitrate,

phosphate and sulphate

Maximum rate
of nitrate Phosphate Sulphate
absorption* absorbed during absorbed during
Period without (jimo\ h~' g"1 5-3 h (/tmol g~l 5-8 h (/imoi g"1
nitrate (d) f. wt root ± s.e.) f. wt root + s.e.) f. wt root ± s.e.)

0 3-4 + 01 2-41+011 0-41 ±003

2-5 7-7 + 0-1 1-36 ±003 0-22 + 002
5 70±01 l-34±0-04 0-23±001

Absorption of nitrate, phosphate and sulphate was measured from aerated solution containing 0-2 mM
potassium nitrate in potassium phosphate-calcium sulphate buffer, pH 6 0 , with the addition of either
~ 100 kBq I"1 [32P]phosphate or ~ 400 kBq I"1 [36S]sulphate. Values of phosphate and sulphate absorption
are means from seven replicate plants. Initial total N concentrations in roots (mg N g~' d. wt): 0 d, 45-9; 2-5 d,
20-4; 5 d, 16-7.
* Rate measured over the 2 h period where the gradient of depletion curve was steepest while the external
nitrate concentration remained > 50/*M, typically 3-0-5-0 h from the start. Means are from four replicate
groups of plants.

We investigated whether the effect of mild N-deficiency on nutrient absorption was

confined to ammonium and nitrate, in view of earlier observations (Lee, 1982) that
enhanced nutrient uptake is restricted to the nutrient which is deficient, or to close
chemical analogues of it. For this purpose nitrate uptake was measured by depletion,
and concurrent uptake of phosphate and sulphate by labelling the potassium phosphate-
calcium sulphate buffer with either [32P]phosphate or [35S]sulphate (but not both in the
22-2
478 Lee and Rudge—Ammonium and Nitrate Absorption by Barley

TABLE 6. Effect of nitrate or potassium deficiencies on the absorption of

ammonium and potassium

Average rate of Potassium absorbed

Composition and ammonium absorption* during 5 h
Period without final pH of (jimo\ h"1 g"1 f. wt (/tmol g"1 f. wt
nitrate (d) uptake solution root + s.e.) root + s.e.)

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Experiment A
0 NoNH4+, pH6-3 4-5 ±0-2
+
0 With NH4 , pH 5 5 3-7 + 0-1 0-8 + 0-04
3 NoNH 4 + ,pH5-6 12-2 + 0-3
3 With NH4+, pH 4-3 10-4 + 0-9 7-6±0-4
3f N o N H / , pH5-6 12 1 ±0 3
3f With NH4+, pH 4-3 11-1+0-9 7-8±0-2
Experiment B
0 With N H 4 + , p H 5-7 4-5 + 0-1 0-9 + 0-07
+
3 With N H 4 , p H 6-OJ 11-1+0-8 11-1 ± 0 - 3
0§ With NH4+, pH 5-4 4-6±0-l 11-2 + 0-2

Absorption of potassium (and ammonium) was measured from 0-2 mM ammonium chloride (where
appropriate) in aerated potassium phosphate-calcium sulphate buffer, initial pH 60, containing also
~ 40-200 kBq I"1 [42K] potassium chloride (additional potassium: 0-08-0-13 mmol I"1). Means are from four
replicate groups of plants. 'Average rate measured while the ammonium concentration was > 50 /m, typically
over the first 4 h. f Sodium sulphate (0-75 mM) added to N-free growth solution for the last 3 d. f Solution
maintained at pH 60 + 005 by dropwise addition of 60 mM sodium hydroxide solution. § Plants grown without
potassium for the last 5 d. Initial potassium concentration: 10-9 mg K g"1 d. wt root, cf. 48-5 mg K g"1 d. wt
root in control plants.

same solution). Nitrogen deficiency, sufficient to double the rate of nitrate absorption,
caused no increase in phosphate or sulphate uptake: instead the rates were both
significantly reduced (Table 5).
We also tested the effect of N-starvation on net uptake of potassium, a putative
analogue of ammonium with regard to its transport properties (see Discussion). In barley
plants, N-starvation stimulated potassium absorption 2-7-fold (in the absence of
ammonium), and ammonium absorption similarly (2-8-fold, in the presence of potassium;
Table 6A). In the presence of ammonium, potassium absorption by N-starved plants
was increased about 10-fold, starting from a smaller N-sufficient control value. Under
these circumstances, the rapid influx of ammonium into N-deficient plants was associated
with an acidification of the external solution from pH 60 to 4-3 over 5 h (cf. a final pH
5-5 for control plants absorbing ammonium). However, this change in the pH of the bulk
solution was not a major factor in enhancing potassium uptake, for an enhancement still
occurred in the absence of ammonium - when the pH of the solution did not fall below
5-8 (Table 6 A) - and also in another experiment when the ammonium-containing
solution was maintained at pH 60 ± 005 (Table 6 B). Neither was accelerated potassium
absorption the result of a decreased sodium concentration in the N-starved plants to
which sodium nitrate had not been supplied, since replacing the sodium as sulphate had
no inhibitory effect (Table 6 A).
By contrast with nitrogen, potassium starvation for 5 d increased subsequent potassium
absorption almost 12-fold (in the presence of ammonium), but ammonium uptake itself
was not affected (Table 6B).
 Lee and Rudge—Ammonium and Nitrate Absorption by Barley 479

DISCUSSION
Effect of N-supply on growth
The Midas barley used in these experiments contained 560-690 /tg N per grain before
germination (1-5—20 per cent N on dry weight basis), placing it in the' low grain nitrogen'
category as denned by Metivier and Dale (1977). After about 6 d, young barley plants
depend on an external supply of N to sustain their maximum rate of growth (Dale,
Felippe and Marriott, 1974), and growth of 'low grain nitrogen' cultivars may be
stimulated by added nitrate even earlier than this (Metivier and Dale, 1977). We found

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that cessation of external N-supply rapidly altered the N-status of the plants. As
previously noted (Vose and Breese, 1964), effects on tissue nitrate concentration were
more severe than those on total N (Table 1), while ammonium concentrations were also
proportionately more stable than those of nitrate (see footnote to Table 1). Greenwood
(1976) concluded that tissue concentrations of total N, and particularly nitrate-N, are
unsatisfactory measures of nitrogen stress, and suggested that for single plants changes
in RGR are more reliable. However, in most of the experiments described here total N,
nitrate-N and ammonium-N concentrations provided the only available indicators of the
nitrogen status of the tissue because - certainly for up to 3 d of nitrate starvation -
nitrogen stress was not severe enough to alter the RGR significantly.
During N-starvation, growth can continue only by the conversion of storage or
relatively inessential forms of N into metabolically essential components to stock the new
tissue being formed. A detailed consideration of this process is easiest in the nitrate-fed
plants, for which more analytical data are available, and where dry-matter accumulation
was exponential during the relevant period (5-10 d from germination) and almost
unaffected by continuation or cessation of the nitrate supply. From the data in Table 1,
and using the ratio of root dry weight to shoot dry weight, it can be calculated that
10-d-old barley plants grown under our conditions with 1-5 mM nitrate as N-source
contained 12-2 mg nitrate N and 39-7 mg non-nitrate N g"1 dry weight of whole plant
(excluding seed remains). Nitrate therefore accounted for more than one-fifth of the total
N in these plants. Similar plants deprived of nitrate for the final 3 d contained 0-9 mg
nitrate N and 25-3 mg non-nitrate N g~x dry weight of whole plant (excluding seed
remains). Thus continued growth without external N severely depleted the intracellular
nitrate reserve, and at the same time the concentration of non-nitrate N in the dry matter
decreased (see above), though over 3 d of starvation metabolically essential components
were clearly not attenuated to the point where dry matter production was significantly
affected. During most of this period (i.e. days 7-10), negligible N would be available from
the endosperm (Metivier and Dale, 1977). Nitrate starvation for 4 d or more depressed
the RGR, however, suggesting that excessive dilution of important components was
beginning to occur.
In a characteristic response to ammonium nutrition (Lewis and Chadwick, 1983; see
also review by Haynes and Goh, 1978), the roots of barley plants grown with ammonium
as N-source contained a higher concentration of total N g"1 dry weight than those of
nitrate-fed plants (Table 2). The free ammonium concentration in these roots
(7-9 fimo\ g"1 fresh weight) was within the range reported for barley grown under broadly
similar conditions (2-4/onol g"1 fresh weight root with 1 mM external ammonium: Lee,
1978; 40-2 /rniol g"1 fresh weight root with 2 mM external ammonium: Lewis, James and
Hewitt, 1982), and represented < 3 per cent of the total N in the root.

Increased capacity for N-absorption following N-starvation

In plants grown on nitrate and then starved of N, stimulation of the potential for
ammonium or nitrate absorption preceded any detectable change in the RGR, and the
480 Lee and Rudge—Ammonium and Nitrate Absorption by Barley
maximum capacity for ammonium or nitrate uptake (g~l f. wt root) was developed before
root growth began to be retarded by N-deficiency (Table 2 B, 3, 4). Likewise, in
ammonium-grown plants, the capacity for additional ammonium absorption was
strongly expressed after 2 d of N-starvation, when root and shoot growth were still
exponential (Table 2 A). The rapidity of this response may reflect the very active
metabolic role of nitrogen; a similar pattern of effects on transport activity anticipating
those on growth has been seen elsewhere, for example in phosphate absorption by
P-deficient tomato and potato plants (Clarkson and Scattergood, 1982; Cogliatti and
Clarkson, 1983), and in sulphate absorption by S-deficient Macroptilium (Clarkson,
Smith and Vanden Berg, 1983). (By contrast, root growth was inhibited quite severely

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before the maximum capacity for phosphate absorption was reached in barley: Clarkson
and Scattergood, 1982; Lefebvre and Glass, 1982.) Comparing experiments on barley
in which all the plants were growing with approximately the same RGR, the timing of
maximum enhancement of uptake capacity has been found to vary widely from one
nutrient to another: it occurred after 3-4 d of N-starvation (see above), but after 12-16 d
of P-starvation (Clarkson and Scattergood, 1982). This may result from differences in
the quantity of reserves (e.g. vacuolar nitrate, vacuolar phosphate) held in nutrient-
sufficient tissue, and in their mobility from the older tissue to new cells formed after the
external nutrient supply is withdrawn (thus affecting the rate at which the nutrient status
of the older tissue declines with time). The rate of development of visible deficiency
symptoms in S-starved barley plants (Lee, 1982) suggests that S may be intermediate
between N and P in this respect.
In the present experiments, we cannot distinguish between changes in plasmalemma
influx or efflux, either of which may in principle vary with nutritional status, but at a
gross level this distinction may be unimportant because it is the difference between the
two rates which determines the availability of nutrient to the plant. Net rates of nitrate
and ammonium uptake were measured under conditions approaching saturation for
'system 1' (Epstein, 1972, p. 132), and changes in F max for net absorption are evidently
involved (see discussion in Lee, 1982). The greatest net rate of nitrate uptake we recorded
was 8-5 /imo\ h"1 g -1 f. wt root from ~ 0-1 mM nitrate solution (Table 3), or an average
of 9-6-101 /tmol h"1 g"1 f. wt root from 1-5 mM solution, measured over 24-25 h
(Table 4). In other studies with intact barley plants under broadly similar external con-
ditions, net rates of nitrate uptake ranged from 1 -8 to 2-1 /miol h"1 g"1 f. wt root after
growth with an abundant nitrate supply (Deane-Drummond, 1982; Lewis and Chadwick,
1983), to 47-7-1-15-8 /miol h"1 g"1 f. wt root in N-starved plants (maximum fully
induced rates: Blevins, Hiatt and Lowe, 1974; Chantarotwong et al., 1976; Deane-
Drummond, 1982). Corresponding values for wheat were 50-9-5 /miol h"1 g"1 f. wt root
(Jackson et al. 1972; Blevins, Barnett and Frost, 1978). Over a short period (0-5 h),
ammonium uptake reached 21-8/tmol h" 1 g"1 f. wt root in N-starved barley plants
(Table 2 A), or an average of 9-6 fimo\ h"1 g"1 f. wt root over 24 h (Table 4). This compares
with a rate of 10-8 /«nol ammonium absorbed h"1 g"1 f. wt root in N-deficient wheat
plants (Jackson et al., 1972), and 2-5-7-2 /miol h~l g"1 f. wt root in intact barley
plants adequately fed with ammonium (Meijer, 1970; Lewis and Chadwick, 1983).
It is clear from Table 4 A that ammonium and nitrate absorption corresponded closely
in groups of plants previously grown on nitrate and then deprived of N for equal periods
of time. Because small variations in ambient conditions may cause slight differences in
growth rate between experiments, it is preferable to compare plants in terms of their
measured initial nitrogen status, rather than by the length of prior N-starvation. This
approach applied to the data in Tables 2 B, 3 and 4 highlights the similarity between
ammonium and nitrate absorption in their response to prior N-starvation. The greatest
net rates of ammonium or nitrate uptake were both achieved in plants whose roots
contained similar initial concentrations of total N (17-7-19-4 mg N g"1 d. wt, Table 2B;
 Lee and Rudge—Ammonium and Nitrate Absorption by Barley 481
19-2-24-7 mg N g"1 d. wt, Table 3). These roots typically contained ~ 10 /tmol nitrate
g"1 f. wt (Tables 3, 4) and ~ 0-8 /tmol ammonium g"1 f. wt at the start (Tables 1, 4A).
Moreover, combined data from several comparable experiments indicated that the
percentage stimulation of uptake of either ammonium or nitrate was closely similar at
a range of intermediate concentrations of total root N (Fig. 2 A).
In ammonium-grown plants, the maximum net rate of ammonium absorption
occurred when the total root N concentration was somewhat higher (28-9-40-3 mg N g"1
d. wt, Table 2 A, cf. Tables 2 B, 3); perhaps fortuitously, the decrease in total N
concentration relative to the unstarved controls was broadly similar in these and in
nitrate-grown plants having maximum N-uptake capacity (~ 30 mg N g"1 d. wt root).

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Note that while total N, nitrate and ammonium analyses refer to the initial status of the
tissue, enough N was taken in during certain experiments to alter the plant composition
appreciably, particularly in N-starved material. For example, in Table 4B the plants
grown without nitrate for 3 d absorbed ~ 670 fig nitrate-N per plant during the 24 h
experiment, a net uptake equal to more than half of the total N content per plant at
the start.

Internal factors controlling N-absorption

The parallel effect of N-starvation on the capacity for net uptake of ammonium
or nitrate - with respect to both magnitude and timing of the response - suggests that
acquisition of either form of N may be influenced by a common underlying factor. A
single transport mechanism operating on both types of ion can presumably be ruled out.
(By implication, precisely parallel control of their efflux rates would also be required.)
What then is the nature of this common factor? Over the long term, net uptake of N
is closely correlated with growth (see review by Clarkson and Hanson, 1980), but in the
present experiments growth clearly did not regulate the capacity for ammonium and
nitrate absorption because this capacity increased (temporarily) during N-starvation in
advance of any change in RGR. Such change in RGR would in any case tend to be in
the opposite direction, that is, an inhibition of growth. Following the model put forward
by Glass (1976) with regard to internal regulation of potassium transport, we assume
that control of nitrate or ammonium uptake is likely to be exerted in the cytosol of root
epidermal and cortical cells, since a control mechanism situated here would be in a
position to regulate both influx and efflux through the plasmalemma, movement into root
cell vacuoles, radial symplastic transport of the ions or their assimilation products (e.g.
glutamine) towards the xylem, and possibly the activity of the enzymes catalysing the
initial stages of assimilation (nitrate reductase, glutamine synthetase). (As a percentage
of the incoming N, transport to the shoot does not seem to be much affected by N-stress,
however; Lee, 1982, p. 434.)
Cytoplasmic nitrate concentration seems unlikely to be a common factor controlling
nitrate and ammonium absorption directly. Although net uptake of nitrate is commonly
suppressed in tissues which contain an abundance of nitrate (Minotti et al., 1969 a;
Jackson el al., 19766; Mackown et al., 1981; Breteler and Nissen, 1982; Talouizte et al.,
1984), this correlation does not invariably occur (Jackson et al., 1976a). Moreover, the
cytoplasmic nitrate pool may be relatively small (Martinoia, Heck and Wiemken, 1981;
Granstedt and Huffaker, 1982; Robin et al., 1983), and the relationship between the
nitrate concentration in this pool in vivo and the averaged - predominantly vacuolar -
nitrate concentration measured by whole-tissue analysis is unknown (see discussion in
Belton et al., 1985). Also it is not clear why cytoplasmic nitrate concentration should
have any direct effect on ammonium transport. Regulation of net ammonium uptake
as a function of N-status occurs in plants grown with ammonium as sole N-source
(Table 2 A), as well as in those grown with nitrate (Tables 2B, 3, 4), and the magnitude
482 Lee and Rudge—Ammonium and Nitrate Absorption by Barley
and timescale of the changes in ammonium uptake capacity which occur as N-stress
develops in the two groups of plants resemble each other fairly closely (Table 2 A, cf.
Tables 2B, 3, 4). Furthermore, external nitrate does not inhibit ammonium absorption
when the two ions are supplied simultaneously even though the nitrate is also taken up
by the tissue and thus passes into, or through, the cytosol (Lycklama, 1963; Fried et al.,
1965; Munn and Jackson, 1978; Breteler and Siegerist, 1984).
Is cytoplasmic ammonium concentration the common factor controlling N-uptake?
Subcellular compartmentation may conceal changes in the concentration of this pool -
which may not necessarily coincide spatially with the rapidly metabolized pool (Fentem,

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Lea and Stewart, 1983; Belton et al., 1985)-and a full understanding must await
methods for studying the compartmentation of ammonium in vivo. At present, it is
difficult to correlate rates of N absorption with average concentrations of ammonium
measured in whole tissues. Thus in our experiments, ammonium concentrations ranged
from ~ 0-8 to l-5fimo\g~1 f. wt root in plants having the maximum capacity for
N-absorption, to 1-7 and 7-9 /rniol g"1 f. wt root in plants grown throughout on nitrate
and ammonium respectively (see footnote to Table 1; Table 2 A). (For comparison, bean
roots with the slowest net uptake of nitrate contained ~ 50 fimo\ ammonium g"1 d. wt
(~ 3 /imo\ g"1 f. wt), accumulated from an external source: Breteler and Siegerist, 1984.)
In the roots of N-starved barley plants, the intracellular ammonium concentration
increased markedly when ammonium was supplied, reaching up to 17 /imo\ g~l f. wt after
5-5 h in plants previously grown on nitrate (Table 2B), and ~ 30/tmol g"1 f. wt after
4 h in plants previously grown on ammonium (Table 2 A); yet in the latter, the net rate
of ammonium uptake over the final 0-5 h (8-8-10-4 fimo\ h"1 g"1 f. wt) was still larger
than in the unstarved controls, which had a lower intracellular ammonium concentration
(8-5 ^mol g"1 f. wet root: Table 2A). (During the final 0-5 h, the external ammonium
concentration was 0-35-0-38 HIM; cf. 0-49 mM in the unstarved controls.)
The view that ammonium is not directly involved in regulating N-uptake is supported
by other results. Inhibition of net absorption of nitrate by external ammonium, supplied
simultaneously, occurs in a wide variety of species, though its detailed aspects are
somewhat variable (see review by Jackson, 1978). A recent investigation (Breteler and
Siegerist, 1984) suggests, however, that this inhibition depends on the flux of N through
the glutamine synthetase/GOGAT pathway, or through its products, rather than on the
intracellular concentration of free ammonium in the tissue, for treatment with methionine
sulphoximine (MSO) or azaserine partially relieved the inhibition of net nitrate uptake
by externally supplied ammonium, even though supplying MSO would be expected to
increase the pool of free ammonium in the roots (as observed during nitrate assimilation
in Datura roots: Probyn and Lewis, 1979). Pre-treating N-starved wheat and bean plants
for 8-18 h with ammonium had little or no effect on the subsequent fully induced net
rate of nitrate uptake in the absence of external ammonium (but no total N analysis was
reported: Tompkins, Jackson and Volk, 1978; Breteler and Siegerist, 1984). Similarly,
in maize grown with ammonium as N-source for 4 d, the fully induced net rate of nitrate
absorption was the same as in plants grown throughout on nitrate (Jackson et al., 1972).
From the latter results it appears that the N-status of the plants, rather than the form
of preceding N-nutrition, was the important factor, as suggested above (Table 2 A, cf.
Tables 2B, 3).
In this context, 'N-status' refers to a property of an N fraction which does not include
nitrate and ammonium. As noted earlier in this discussion, the concentration of
non-nitrate N in the dry matter of nitrate-grown plants declined by 36 per cent during
the first 3 d of N-starvation, when the capacity for nitrate transport was increasing
without change in RGR. (Ammonium-N forms a negligible proportion, < 1 per cent,
of the total N in nitrate-grown plants.) From considerations of their function, it is likely
that the concentrations of membrane proteins, nitrogenous phospholipids, nucleic acids,
 Lee and Rudge—Ammonium and Nitrate Absorption by Barley 483
nucleoproteins and certain 'soluble' proteins (e.g. glycolytic and Krebs-cycle enzymes)
will remain fairly constant, per unit of dry matter produced, in a given tissue over a range
of RGRs, whether growth is limited by N-supply or not (cf. stability of phospholipid-P
concentration during variations in P-supply: Chisholm et al., 1981). Immediate control
of net uptake of N is probably exerted through some component(s) in the lower molecular
weight range (see for example references in Syrett, 1981, and Breteler and Arnozis, 1985),
but no consistent pattern could be discerned in the effects of exogenous amino acids on
nitrate absorption by bean roots (Breteler and Arnozis, 1985). The situation is
complicated by subcellular compartmentation of many such compounds having possible

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regulatory roles (Fentem et al., 1983; see also reviews by Oaks and Bidwell, 1970; Miflin
and Lea, 1980), and further progress in this area must await techniques for measuring,
and preferably also for varying experimentally, concentrations of individual nitrogenous
compounds in specific compartments of the cell.
The additional transport capacity for nitrate which develops during mild N-stress is
selective against two other major anionic nutrients, phosphate and sulphate (Table 5).
Parallel examples of selectivity were noted during previous studies of nutrient stress in
barley (Lee, 1982). Conversely, it is reasonable to assume that net uptake of the nitrate
analogue, chlorate, would be stimulated in N-deficient plants (Deane-Drummond and
Glass, 1982). Compared with the unstarved controls, plants which had been deprived
of N for 2-5 d absorbed an additional 22-6 /<equiv nitrate g"1 f. wt root over 5-5 h when
nitrate was re-supplied (calculated from average net rates of nitrate uptake by the plants
in Table 5); this far exceeded the decrease in phosphate plus sulphate absorption by the
same plants during the same period (~ 1 -6 /tequiv g"1 f. wt root). Charge balance during
rapid nitrate uptake may have been achieved by an accompanying influx of protons or
efflux of hydroxyl ions, the effect being partially hidden by the phosphate buffer in the
external solution. It is also likely that potassium absorption was accelerated (Minotti
et al., 1969a; Jackson et al., 1976ft; Blevins et al., 1978).
Potassium and ammonium behave in a broadly similar way during certain transport
processes (see review by Haynes and Goh, 1978), although it is not clear to what extent
they should be considered strict analogues in this context. Ammonium inhibits potassium
uptake markedly in many species including wheat (Tromp, 1962), barley (Bange, Tromp
and Henkes, 1965; Meijer, 1970), maize (Rufty, Jackson and Raper, 1982) and tobacco
(Scherer, Mackown and Leggett, 1984), but the converse interaction is smaller and
sometimes negligible (e.g. wheat and maize, see references above). The relatively minor
effect of potassium on ammonium absorption by tobacco plants was characterized as
a mixed competitive/non-competitive inhibition (Scherer et al., 1984). In our experiments,
potassium and ammonium both responded similarly in that their net rates of uptake were
stimulated to the same extent in N-starved roots (Table 6 A). In contrast, Tromp (1962)
found that potassium absorption was suppressed during rapid ammonium absorption
by N-deficient plants. However, potassium starvation does not produce the same effect
as N-starvation on the transport of ammonium, since there was clear selectivity in favour
of enhanced potassium absorption by K-deficient barley plants (Table 3 B), as seen also
in wheat (Tromp, 1962).
In summary, the effects of N-starvation on nutrient absorption by young barley plants
show, on a compressed timescale, several similarities with the effects of P- and S-starvation
(Lee, 1982), notably the increase in Kmax for net uptake of the nutrient, and the selectivity
favouring nitrate absorption over that of other anionic nutrients. But there are also
certain differences. The intensity of the response tends to be smaller, particularly
compared with the effects produced by S-stress (Lee, 1982; Clarkson et al., 1983), and
there is an important feature not seen in connection with P- and S-starvation, namely
that N-starvation increases the capacity for net uptake of N in two different states of
oxidation and charge: the anionic nitrate and the cationic ammonium. Clearly an
484 Lee and Rudge—Ammonium and Nitrate Absorption by Barley
analogous situation cannot arise with respect to P, which is absorbed only as the
orthophosphate anion, but it is interesting to note that, at least in higher plants,
absorption of reduced forms of S is not accelerated by mild S-deficiency. Thus although
L-cysteine will serve as an effective source of S through intracellular conversion to
sulphate, the absorption of L-cysteine was not stimulated (in contrast to that of sulphate)
in S-deficient Macroptilium (Clarkson et al., 1983).
From an evolutionary viewpoint, the differing behaviour of N and S in this respect
may possibly be explained by the fact that reduced sulphur compounds are not available
as a source of S for most higher plants under field conditions (except perhaps to parasitic

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or saprophytic species). In soils, reduced sulphur compounds occur in significant
quantities only when aeration is severely restricted, for example by waterlogging. On the
other hand, the soil solution typically contains both nitrate and ammonium ions in
proportions which vary with the type of soil and with the season (Haynes and Goh, 1978),
though in nature the presence of ammonium alone is probably rare (Reisenauer, 1978).
Hence an ability to expand the capacity for N-absorption, irrespective of which form
predominates in the soil solution at the time, may be physiologically advantageous in
allowing changes in growth rate caused by variations in the above-ground environment
to be matched by corresponding changes in the rate at which N is acquired.

ACKNOWLEDGEMENTS

We are grateful to Mrs S. A. Lawther, Mr P. D. Robertson and Mr M. G. Johnson for

the nitrate, ammonium and l5 N analyses.

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