J. Plant Nutr. Soil Sci.

2016, 000, 19

DOI: 10.1002/jpln.201500625

Small amounts of ammonium (NH

4 ) can increase growth of maize
(Zea mays)
Jessey George1,2, Luke Holtham1,2, Kasra Sabermanesh1,2, Sigrid Heuer1,2, Mark Tester1,3, Darren Plett1,2*,
and Trevor Garnett1,2
1

School of Agriculture, Food and Wine, Waite Research Institute, University of Adelaide, Urrbrae, SA 5064, Australia
Australian Centre for Plant Functional Genomics, Waite Research Institute, Urrbrae, SA 5064, Australia
3 King Abdullah University of Science and Technology, Center for Desert Agriculture, Thuwal, Kingdom of Saudi Arabia, 23955-6900
2

Abstract

Nitrate (NO
3 ) and ammonium (NH4 ) are the predominant forms of nitrogen (N) available to
plants in agricultural soils. Nitrate concentrations are generally ten times higher than those of
NH
4 and this ratio is consistent across a wide range of soil types. The possible contribution of
these small concentrations of NH
4 to the overall N budget of crop plants is often overlooked. In
this study the importance of this for the growth and nitrogen budget of maize (Zea mays L.) was
investigated, using agriculturally relevant concentrations of NH
4 . Maize inbred line B73 was
grown hydroponically for 30 d at low (0.5 mM) and sufficient (2.5 mM) levels of NO
3 . Ammonium
 levels, addition of NH
was added at 0.05 mM and 0.25 mM to both levels of NO
.
At
low
NO
4
3
3
was found to improve the growth of maize plants. This increased plant growth was accompanied
by an increase in total N uptake, as well as total phosphorus, sulphur and other micronutrients in
the shoot. Ammonium influx was higher than NO
3 influx for all the plants and decreased as the
total N in the nutrient medium increased. This study shows that agriculturally relevant proportions

of NH
4 supplied in addition to NO3 can increase growth of maize.

Key words: ammonium / nitrate / ion fluxes / N uptake / N budget

Accepted August 19, 2016

1 Introduction
Nitrogen (N) is one of the major nutrients required by plants
with N limitation commonly leading to reduction in both growth
and yield. Plants absorb N mainly in the form of nitrate (NO
3)
and ammonium (NH
4 ) from the soil solution (Glass et al.,
2002). Nitrate is the dominant form present in agricultural
soils and, hence, is the focus of most research on N uptake
(Marschner, 2011). Although the NH
4 concentration in the
soil solution is small ( 10%) compared to NO
3 , this ratio is
consistent in most agricultural soils (Miller and Cramer, 2005)
and could make a significant contribution to plant N uptake.
Previous studies have demonstrated that a combination of

NO
3 and NH4 is beneficial for plant growth (Haynes and
Goh, 1978; Bloom et al., 1993). This has also been shown in
maize (Schrader et al., 1972; Smiciklas and Below, 1992).
However, these studies were conducted using proportions of
NH
4 greater than 25% (Schrader et al., 1972), which is much
higher than the concentrations typically present in agricultural
soils. Cox and Reisenauer (1973) used small amounts of

NH
4 relative to NO3 and found that, even at these low proportions, growth of wheat was stimulated. Therefore, it is
important to understand whether agriculturally relevant proportions of NH
4 increase growth of maize and, if there is any
increase, why is this so.

There are a number of possible reasons for the increase in

plant growth that occurs with a mixture of NO

3 and NH4 .

Nitrate first has to be reduced to nitrite (NO2 ) in the cytosol;

the NO
2 then enters plastids where it is converted to NH4 ,
from which amino acids are formed (Bloom et al., 1992). This
additional processing means that NO
3 assimilation is a
more energy-intensive process [requiring 12 adenosine triphosphate (ATP) molecules] compared to NH
4 assimilation
(requiring two ATPs) (Schrader et al., 1972; Clarkson, 1985).
On this basis, it may be that energy conservation is one factor
underlying the increased plant growth observed with small
amounts of NH
4.
Another reason for the increased plant growth may be related

to the higher uptake capacity of NH
4 relative to NO3 when
both N sources are present. Previous studies have shown that
when both N forms are available, plants take up NH
4 preferentially over NO
(Hatch
and
Macduff,
1991;
Gazzarrini
et al.,
3
1999; Glass et al., 2002), and that the total N uptake of these
plants is increased. The reason for preferential uptake of NH
4
may be due to the faster assimilation of NH
4 in the roots (Lee
et al., 1992) by the enzymes cytosolic glutamate synthetase
(GS1) and glutamate synthase (GOGAT) resulting in a more
rapid incorporation of inorganic N into organic N.
This positive growth effect of NH
4 could also be due to effects
on other aspects of plant nutrition, given that in some cases

* Correspondence: D. Plett; e-mail: darren.plett@acpfg.com.au

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George, Holtham, Sabermanesh, Heuer, Tester, Plett, Garnett

NH
4 can improve the phosphorus (P), sulphur (S) (Kirkby,
1968; Flores et al., 2001), and micronutrient (Riley and Barber,
1971; Thomson et al., 1993; Jeong and Lee, 1996) nutrition of
plants. The uptake of NO
3 by plants can enhance the absorption of cations such as K+, Mg2+, and Ca2+, but can lead to
slower uptake of P, S, and some trace elements (Jackson and
Williams, 1968). Iron (Fe) deficiency has been observed in
plants grown solely with NO
3 compared to plants grown with
 (Zou et al., 2001). On the other hand,
both NH
and
NO
4
3
when NH
4 is supplied to plants it can increase the uptake of
Fe from the nutrient solution, and the re-mobilisation of Fe inside the plant (Marschner et al., 1987; Zou et al., 2001).
The aim of this study was to compare and quantify the effect
of low and high levels of NH
4 on maize growth at both low
and sufficient levels of N. This study looked at the effect of

small levels of NH
4 on total N uptake, C : N ratio, NH4 and

NO3 influxes and on macro and micronutrient uptake. Maize
inbred line B73 was used as it is the source of the maize
reference genome and has been used in a large number of
physiological studies (Agrama et al., 1999; Martin et al.,
2006). In this manuscript, we report the effect of agriculturally
relevant proportions of NH
4 on the maize inbred line when
provided along with NO
.
3

2 Material and methods

2.1 Plant material and growth conditions
Seeds of maize (Zea mays L. inbred line B73) were soaked
overnight in water, placed on filter paper moistened with
0.5 mM CaCl2 and placed in an incubator at 28C. The germinated seedlings were transferred into a climate-controlled
growth chamber providing a day/night temperature of 26/22C
and a photoperiod of 14 h. The photon flux density in the
growth chamber was approximately 450 mmol m2 s1 at average leaf height. Plants were grown on mesh collars in tubes
as outlined in Garnett et al. (2013). The N treatments con

tained 0.5 mM NO
3 + 0.05 mM NH4 (LN+LA), 0.5 mM NO3 +

0.25 mM NH4 (LN+HA), 2.5 mM NO

+
0.05
mM
NH
4
3


(SN+LA), and 2.5 mM NO3 + 0.25 mM NH4 (SN+HA). All
these treatments contained different N concentrations. A second experiment was conducted in a similar manner, but the N
concentration remained the same in both treatments of this
experiment which consisted of 0.75 mM NO
3 alone (LN+0A)

and 0.5 mM NO

3 + 0.25 mM NH4 (LN+HA). Table 1 shows
the various treatments in the two experiments. Johnsons
modified nutrient solution (Johnson et al., 1957) was used
containing (in mM) 0.8 K, 0.1 Ca, 0.5 Mg, 1 S, 0.5 P, and
(in mM) 2 Mn, 2 Zn, 25 B, 0.5 Cu, 0.5 Mo, and 200 Fe (as
Fe-EDTA and Fe-EDDHA). Iron was supplemented twice
weekly with the addition of Fe(NH4)2(SO4)2 6 H2O

(8 mg L1), and this was the NH

4 source for 0.05 mM NH4 in
all the treatments. (NH4)2SO4 was used as the NH
supple4

ment to provide the extra 0.2 mM NH

4 in the 0.25 mM NH4
treatments. Solution pH was monitored daily and maintained
between 5.9 and 6.0 by adding either 1 M H2SO4 or
1 M NaOH. Nitrate and NH
4 concentrations were monitored

in the solutions using NO

3 and NH4 electrodes (TPS,
Springwood, Australia) and maintained at the target concen-

2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

J. Plant Nutr. Soil Sci. 2016, 000, 19

Table 1: The NO

3 and NH4 treatments in the experiments.

Experiment

Treatments
NO
3 (mM)

NH
4 (mM)

Experiment 1

LN+LA
LN+HA
SN+LA
SN+HA

0.50
0.50
2.50
2.50

0.05
0.25
0.05
0.25

Experiment 2

LN+0A
LN+HA

0.75
0.50

0.25

tration 5%. Nutrient solutions were replaced 23 d after

emergence (DAE) and the plants were grown for 30 d.

2.2 Plant biomass and chemical analysis

In Experiment 1, plant biomass was measured at 10, 17, 23,
and 30 DAE. In Experiment 2, plants were measured at
23 DAE. In both the experiments roots and shoots were separated, and the roots blotted with paper towel before weighing
to obtain fresh biomass. Plant parts were then dried at 40C
for 7 d to obtain dry weights. Root : shoot ratio was calculated
using the dry weights. Dry matter was ground to a fine powder
and shoot tissues were analysed for N and C using a mass
spectrometer (Sercon, Cheshire, UK). Shoot macro- and micronutrient contents were determined following acid digestion
using inductively coupled plasma-optical emission spectrometry (ICP-OES; ARL 3580B, ARL, Lausanne, Switzerland).

2.3 Influx measurements

Unidirectional fluxes into roots were measured at 23 DAE in

Experiment 1 using 15N-labelled NO

3 and NH4 . On the day
of sampling, plants were transferred to a controlled environment room with matching growth conditions. The whole root
system of the plants was then moved to a nutrient solution
14NH for 5 min, and subsecontaining 100 mM 14NO
4
3 or
quently to flux solutions containing either 100 mm NO
3 or
15N (15N 10%) for 10 min. The whole roots
NH
4 labelled with

were then rinsed in unlabelled 100 mM NO

3 or NH4 solution
15
for 2 min to wash the N from the root surface and apoplast.
As described by Kronzucker et al. (1995), a flux timing of
10 min was chosen to minimise any N efflux or transport to
shoots. Roots were then separated from shoots, blotted and
weighed. Plant parts were dried in an oven at 40C for 7 d.
After measuring the dry matter content, the roots were ground
to a fine powder and the total N and 15N in plant samples
were determined with an isotope ratio mass spectrometer
(Sercon, Cheshire, UK). Unidirectional NO
3 influx was calculated based on 15N content of the root.

2.4 Statistical analysis

Statistical analyses of biomass, total N, fluxes and macroand micronutrient contents were carried out using two-way
analysis of variance (ANOVA) in Graph Pad Prism software
(GraphPad Software, Inc).

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J. Plant Nutr. Soil Sci. 2016, 000, 19

3 Results
3.1 Plant biomass
The shoot biomass of plants grown in low N was greater (at
30 DAE) with added NH
4 in the LN+HA treatment; however,
when grown with sufficient N, the plants showed no response
to NH
4 (Fig. 1A). Plants grown in higher concentration of
NH
4 showed no change in root biomass regardless of N
treatment (Fig. 1B), except on 23 DAE, which had a higher
root biomass in SN+LA compared to SN+HA. Plants grown in
LN+LA and LN+HA had a higher root : shoot ratio compared
to plants in both sufficient N treatments at 10 DAE (Fig. 1C).
Thereafter, this ratio decreased for plants in all treatments
and at final harvest, a decrease was observed in the root:
shoot of plants in LN+HA compared to LN+LA and both sufficient N treatments.

to LN+LA in Experiment 1 was due to the extra N contributed

by the added NH
4 or the presence of high NH4 in LN+HA.

Plants were tested for their response to NH4 at low N levels,

but the total N level was kept constant at 0.75 mM using NO
3

alone in one treatment, and 0.5 mM NO

3 and 0.25 mM NH4
in the other treatment. It was observed that plants grown in a

mixture of NO
3 and NH4 (LN+HA) had higher shoot dry matter (Fig. 2A) than plants grown in NO
3 alone (LN+0A). No difference in root dry matter was observed between treatments
(Fig. 2B). The root : shoot ratio of plants was higher with NH
4
at 10 DAE (Fig. 2C). Thereafter, the root : shoot ratio de
creased in plants treated with NH4 and was lower at 20 and
23 DAE than for plants grown with NO
3 alone.

3.2 Tissue N concentration and total N

Maize plants grown in SN+LA had lower N concentration in
their shoots at 17 DAE compared to all other treatments
(Fig. 3A). However, at 30 DAE, the tissue N concentration

Root : shoot ratio

Root : shoot ratio

Root DM / g

Root DM / g

Shoot DM / g

Shoot DM / g

Experiment 2 was conducted to determine whether the observed higher biomass of maize plants in LN+HA compared

Effect of ammonium on maize growth 3

Days after emergence (DAE)

Figure 1: Shoot dry matter, root dry matter, and root : shoot ratio of
maize inbred line B73 (A, B, C) grown in 0.5 mM NO
3 + 0.05 mM



NH
4 (LN+LA), 0.5 mM NO3 + 0.25 mM NH4 , (LN+HA), 2.5 mM NO3
 + 0.25 mM NH
+ 0.05 mM NH
(SN+LA),
and
2.5
mM
NO
4
4
3
(SN+HA). Values are means SE, n = 6. Significant differences for
individual harvest days are represented by different letters (P 5%).

2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Days after emergence (DAE)

Figure 2: Shoot dry matter (A), root dry matter (B), and root : shoot
ratio (C) of maize inbred line B73 grown in 0.75 mM NO
3 (LN+0A)

and 0.5 mM NO

+
0.25
mM
NH
(LN+HA).
Values
are
means
SE,
4
3
n = 6. Significant differences between treatments on each day of harvest with *P 5%).

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George, Holtham, Sabermanesh, Heuer, Tester, Plett, Garnett

was higher for plants grown in LN+HA compared to plants in

LN+LA; no difference was observed with NH
4 for either sufficient N treatment (Fig. 3A). Similarly, maize plants grown in
LN+HA had higher total N and net uptake than LN+LA on
both 17 and 30 DAE, while there was no difference between
SN+LA and SN+HA (Figs. 3B, C). At the two harvests, plants
grown in LN+LA had higher C : N ratio in their shoots,
compared to LN+HA (Fig. 3D).
Similar to Experiment 1, in Experiment 2, plants grown in

NO
3 and NH4 (LN+HA) had a higher shoot N concentration
(Fig. 4A) than plants grown in NO
3 alone (LN+0A). Total N
and net uptake relative to root dry matter content also showed
an increase in LN+HA compared to LN+0A (Fig. 4B, C). No
difference in the C : N ratio of plants was observed between
the two treatments.

3.3 Nitrate and ammonium influx

High affinity influx was measured for plants in Experiment 1 at

23 DAE using 15N-labelled 100 mM NH
4 or NO3 solutions.

The NH4 influx of all the plants was several fold higher in all
the treatment compared to their corresponding NO
3 influx
(Fig. 5). The NO
influx
was
lower
only
for
maize
plants
3
grown in SN+HA (Fig. 5). In comparison, NH
influx
de4
creased as N level in the medium increased.

3.4 Macro- and micronutrient uptake

of most micronutrients in maize, except for manganese (Mn;

Fig. 6B). Of all the micronutrients, Fe uptake showed the
greatest increase for plants in LN+HA, compared to LN+LA
(Fig. 6B).
Plants grown with NH
4 had higher concentrations of P and S
compared to plants grown in NO
3 alone in Experiment 2
(Fig. 7A). However, a decrease in Ca concentration was observed in plants supplied with some NH
4 (LN+HA). The tissue concentration of all micronutrients increased when plants

were grown in a mixture of NO

3 and NH4 , with the exception
of Mn and Zn, which both decreased (Fig. 7B).

4 Discussion
This study investigated the effect of agriculturally relevant

proportions of NH
4 and NO3 on the growth of maize. Maize
plants were grown in two levels of NO
3 , either low (0.5 mM)
or sufficient (2.5 mM) and with two levels of NH
4 (0.05 mM or
0.25 mM) for each NO
treatment
(Table
1).
These
results in3
dicate that shoot biomass of maize plants was increased

when more NH
4 was added to the low NO3 treatment
(Fig. 1A). In this experiment, the extra total N provided in the
added NH
4 may have contributed to the increased biomass
of the plants. However, the increased biomass in the low

NO
3 with high-NH4 treatment compared to NO3 -only treatment in Experiment 2 suggests that the increase in plant

growth observed in low-NO

3 with high-NH4 treatment in Experiment 1 was due not to the increase in total N (Fig. 2A),
but specifically to the added NH
4 . Other studies have also
shown increases in grain yield and whole-shoot N content

when a mixture of NO

3 and NH4 were supplied to maize (Below and Gentry, 1987). These results support those found by

C : N ratio

Net uptake N / g N g

root DW

Total N / g

Shoot N%

In Experiment 1, shoot sulphur (S) and phosphorus (P) concentrations in maize plants grown in LN+HA were higher than
for those grown in LN+LA (Fig. 6A). Increasing NH
4 concentration at low NO
levels
increased
the
tissue
concentration
3

J. Plant Nutr. Soil Sci. 2016, 000, 19

Days after emergence (DAE)

Days after emergence (DAE)

Figure 3: Shoot N concentration, total N content, net uptake, and C : N ratio of the maize inbred line B73 (A, B,



C, D) grown in 0.5 mM NO
3 + 0.05 mM NH4 (LN+LA), 0.5 mM NO3 + 0.25 mM NH4 , (LN+HA), 2.5 mM NO3
 + 0.25 mM NH (SN+HA). Values are means SE, n = 6. Signifi+ 0.05 mM NH
(SN+LA),
and
2.5
mM
NO
4
4
3
cant differences are represented by different letters for each group of bars at individual harvest days (P 5%).

2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Total N / g

Effect of ammonium on maize growth 5

C : N ratio

Net uptake N / g N g1 root DW

Shoot N%

J. Plant Nutr. Soil Sci. 2016, 000, 19

Influx / mol (g DW h)1

Figure 4: Shoot N concentration (A) and net uptake (B) of the maize inbred line B73 grown in 0.75 mM NO
3 (LN+0A) and

0.5 mM NO
3 + 0.25 mM NH4 (LN+HA) on 23 DAE. Values are means SE (n = 8). Significant differences between treatments with *P 5%).

Figure 5: Ammonium and nitrate influxes measured at 100 mM 15N

concentration in maize inbred line B 73 (A) grown in 0.5 mM NO
3 +
 + 0.25 mM NH , (LN+HA),
0.05 mM NH
(LN+LA),
0.5
mM
NO
4
4
3


2.5 mM NO
3 + 0.05 mM NH4 (SN+LA), and 2.5 mM NO3 + 0.25 mM

NH4 (SN+HA) on 23 DAE. Values are means SE, n = 4. Significant

differences are represented by different letters for each group of bars
(P 5%).

Cox and Reisenauer (1973) where 10% NH

4 as a proportion
of total N stimulated growth of wheat.
A number of hypotheses have been put forward to explain the

increase in plant biomass with a mixture of NO

3 and NH4 .
These hypotheses relate to: (1) the increased growth due to
the effect of NH
4 on the C : N ratio, (2) increased total N up
take, (3) differing NH
4 and NO3 uptake capacities, and (4)

2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

enhanced macro and micro nutrient nutrition. Nitrogen assimilation is very closely related to C metabolism because C skeletons produced by photosynthesis are required for amino
acid synthesis (Kaiser and Forster, 1989; Pace et al., 1990).
The decreased C : N ratio and higher N concentration of

plants in low-NO
3 with high-NH4 is consistent with lower N
assimilation costs with added NH
4 compared to plants in low

NO
3 with low NH4 treatment. The lower root : shoot of NH4 treated plants throughout the growth period, except on day 10
in Experiment 2, suggests that less C is required for root proliferation in comparison to the increased shoot growth in
these plants. Increased biomass allocation to the shoot
compared to root increases carbon assimilation capacity and
plant relative growth rate (Van der Werf, 1996). This may explain the increased growth of plants in the low-N treatments
following the addition of NH
4.
Another hypothesis is that increased plant growth may result
from improved N status with the addition of NH
4 . This may be
due to a higher uptake capacity of NH
4 , which would result in
faster incorporation of N into an organic form and a corresponding increase in plant growth with high NH
4 concentration. For tomato plants provided with various ratios of radio
labeled NO
3 and NH4 , Glass et al. (2002) reported that 50%
of total N was from NH
4 even though it constituted only 10%
of the total N in the growth medium. Moreover, the extra NH
4
assimilated in the roots may have resulted in increased translocation of N assimilatesmainly amino acidsto the shoots,
resulting in increased shoot growth. This hypothesis has also
been proposed in earlier studies on maize (Alexander et al.,
1991; Gentry, 1992; Cramer and Lewis, 1993) and sorghum
(Lewis et al., 1982), in which the higher concentration of NH
4
in the growth solution was found to increase total N uptake.

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George, Holtham, Sabermanesh, Heuer, Tester, Plett, Garnett

Macronutrients / mg kg
1

Micronutrients / mg kg
Figure 6: Macronutrient (A) and micronutrient (B) concentrations of the shoots of maize


inbred lineB73 grown in 0.5 mM NO
3 + 0.05 mM NH4 (LN+LA), 0.5 mM NO3 + 0.25 mM


NH4 , (LN+HA), 2.5 mM NO3 + 0.05 mM NH4 (SN+LA), and 2.5 mM NO
3 + 0.25 mM
NH
4 (SN+HA) on 30 DAE. Values are mean SE, n = 6. Significant differences are represented by different letters for each group of bars (P 5%).

Net N uptake increased with the addition of NH

4 to the nutrient solution, suggesting that NH
4 uptake capacity is higher
compared to NO
3 uptake capacity in maize plants. The two

and
NH
main NO
4 uptake systems in plants are the satura3
ble high affinity transport system (HATS) and non-saturable
low affinity transport system (LATS; Forde, 2000). In the cur
rent study, the influx via HATS of both NH
4 and NO3 played a
major role in the N uptake response of maize to N demand
(Malagoli et al., 2004; Garnett et al., 2013). The finding here

that NH
4 influx was higher than NO3 influx is consistent with
previous work (Lee and Rudge, 1986; Teyker et al., 1988;
Hole et al., 1990; Glass et al., 2002). Furthermore, it was
found that, even when the concentration of NO
3 is ten times
higher than that of NH
4 , plants have the tendency to absorb

NH
4 more rapidly than NO3 (Gessler et al., 1998).
The NH
4 influx of plants decreased as N levels in the growth
solution increased, indicating that these plants alter their influx based on total N supply. This result is expected as a number of studies show that nutrient deprivation augments the
transport capacity of the deficient ion (Clarkson et al., 1983;

2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

J. Plant Nutr. Soil Sci. 2016, 000, 19

Cogliatti and Clarkson, 1983). All these reports indicate that the response is specific
to the deficient nutrient (Lee, 1982), which
suggests that the finding by Lee and
Rudge (1986), that deprivation of NO
3
augments the transport system for NH
4 , is
unusual. However, the results of the current
study support those of Lee and Rudge
(1986) in that a higher NH
4 uptake capacity
is observed for plants that were grown in
low NO
3 . Nitrate uptake capacity was lower only for plants with sufficient N and high
NH
4 and was unaffected by total N in the
medium. However, the NH
4 uptake capacity of maize plants decreased as the N
in the nutrient medium was increased.

When plants were grown in low-NO

3 with

high-NH
4 treatment (the proportion of NO3

and NH
being
2
:
1)
compared
to
low-NO
4
3

with low NH4 -treatment, both the P and S

concentrations in maize plants increased with
no observed difference in K concentration.
Similar results were obtained in tomato plants
under mixed N nutrition in which the PO3
4
and SO2
4 were increased when the ratio of

NO
3 and NH4 was 12 : 2 and 10 : 4 (Flores et
al., 2001). Earlier studies suggested that enhanced P uptake in plants supplied with NH
4
may be due to acidification at the root surface
caused by absorption and assimilation of
NH
4 (Miller et al., 1970). Recent research indicated the involvement of plasma membrane H+ ATPase in the enhanced uptake of
P by rice roots supplemented with NH
4
(Zeng et al., 2012). The authors suggested
that plasma membrane H+ ATPase activity is
increased by low pH resulting from NH
4 nutrition which facilitates uptake.

The concentrations of the micro nutrients Fe, Cu, Mo, and Zn

increased with increasing NH
4 at both low and high N levels.
Similar results have been demonstrated in beans (Thomson
et al., 1993) and the increased uptake thought to be due to
NH
4 -induced acidification increased micronutrient availability
(Sarkar and Wyn Jones, 1982). Even though the pH of the
bulk nutrient solution was maintained at 5.9, the effect of
changed apoplastic pH cannot be ruled out as playing a role
in increased nutrient availability and hence uptake (Gerendas
et al., 1993; Mengel et al., 1994; Muhling and Lauchli, 2001).
However, as none of the macro- or micronutrients was in the
deficiency range (Reuter et al., 1997), it is unlikely the observed differences caused differences in growth.
Another important factor that cannot be ruled out for the bene
ficial effect of NH
4 nutrition is that assimilation of NH4 requires less energy than assimilation of NO
and
it
is
assimi3
lated in the roots (Bloom et al., 1992). Although we did not
directly evaluate this factor in our study, the energy savings

associated with assimilating NH
4 rather than additional NO3
may have contributed to the growth increase we observed.

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Macronutrients / mg kg1

J. Plant Nutr. Soil Sci. 2016, 000, 19

Effect of ammonium on maize growth 7


and sufficient N levels and found that adding NH
4 with NO3
at low N levels improved growth in maize, with a corresponding increase in the total N content and also a higher uptake of
several essential nutrients. The results obtained here are consistent with NH
4 stimulating growth which could potentially be
explained by lower energy requirements or improved macroand micronutrient nutrition. Further studies are being carried
out to more fully understand the mechanisms underlying this
stimulation of growth.

Acknowledgments

Micronutrients / mg kg1

The authors would like to acknowledge the technical assistance provided by research and technical staff at Australian
Centre for Plant Functional Genomics (ACPFG) and Plant
Research Centre in the University of Adelaide. This study
was funded by the Australian Centre for Plant Functional Genomics (ACPFG), the Grains Research and Development
Corporation (GRDC), and Australian Research Council Linkage Grant (LP130101055).

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Figure 7: Macronutrient (A) and micronutrient (B) concentrations of

the shoots of maize inbred line B73 grown in 0.75 mM NO
3 (LN+0A)

and 0.5 mM NO

+
0.25
mM
NH
(LN+HA)
on
23
DAE
Values
are
4
3
mean SE, n = 8. Significant differences between treatments within
each group of bars with *P 5%).

Studies in the past have also indicated that there is contradicting effect of N forms on phytohormones namely cytokinins
which are decreased when NH
4 is supplied (Walch-Liu et al.,
2000). Ammonium induced perturbations of osmotic homeostasis (Chaillou et al., 1991) is another possibility that cannot
be ruled out for the increased plant growth when NH
4 and
NO
3 are simultaneously supplied. However, these studies

used high ratios of NH
4 and NO3 , so it is difficult to relate to
effects of small NH
concentrations.
4

5 Conclusions
Previous studies have shown that plants grow better with a


mixture of NO
3 and NH4 rather than NO3 as the sole N
source, but these studies used concentrations of NH
4 that
are higher than those usually found in agricultural soils. This
study investigated the contribution of agriculturally available
concentrations of NH
4 when supplied together with low N

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