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ORIGINAL ARTICLE
OsAMT1.3 expression alters rice ammonium uptake kinetics
and root morphology
Leandro Martins Ferreira
1
•Vinicius Miranda de Souza
1
•
Orlando Carlos Huertas Tavares
1
•Everaldo Zonta
1
•Claudete Santa-Catarina
3
•
Sonia Regina de Souza
2
•Manlio Silvestre Fernandes
1
•Leandro Azevedo Santos
1
Received: 26 January 2015 / Accepted: 20 June 2015
ÓKorean Society for Plant Biotechnology and Springer Japan 2015
Abstract High-affinity ammonium transporters (AMT1)
are responsible for ammonium (NH
4
?
) acquisition and/or
perception in the micromolar range, and their expressions
can be differentially regulated by nitrogen (N) availability.
The present study characterised the functions of the rice
(Oryza sativa)OsAMT1.3 transporter to understand its
contribution to NH
4
?
acquisition and plant adaptation to
environments with low N availability. Transgenic rice
plants were obtained to study the activity of the OsAMT1.3
promoter (P
OsAMT1.3
:GFP:GUS) and the overexpression of
the OsAMT1.3 gene (UBIL:OsAMT1.3:3xHA) in plants.
The OsAMT1.3 promoter activity was induced strongly in
the absence of N and occurred primarily in the zones of
lateral root emission and root tips. Anatomical sections of
the segment of root tips and the middle third showed a
differential pattern of OsAMT1.3 activity. Analysis of the
OsAMT1.1–1.3 transporter expression profiles indicated
that overexpression of OsAMT1.3 positively affected
OsAMT1.2 expression. When subjected to a low N supply,
plants overexpressing OsAMT1.3 showed lower K
M
and
C
min
values. Additionally, these lines showed longer roots
with a higher area, volume, and number of tips. The data
suggested that OsAMT1.3 is involved in the ability of rice
plants to adapt to low NH
4
?
supplies.
Keywords Oryza sativa L. Nitrogen Ammonium
transporter qRT-PCR
Introduction
Nitrogen (N) is an essential element for plants and is the
most limiting element for crop productivity and cereal
grain quality (Bu et al. 2011). The current high rice pro-
ductivities became possible partly because of the intensive
use of fertilisers, primarily N fertilisers. Approximately
110 million tons of N fertilisers are applied annually
worldwide, at high cost (FAO 2012). Additionally, this
excess application may be associated with severe envi-
ronmental damage (Mulvaney et al. 2008).
Plants absorb N preferentially as ammonium (NH
4
?
) and
nitrate (NO
3
-
). Under anaerobiosis, NH
4
?
is the main form
of N available to plants (Funayama et al. 2013). However,
excess NH
4
?
absorption can be toxic (Britto et al. 2001),
and the absorption and metabolism of this nutrient is highly
regulated in plants (Sonoda et al. 2003a,b).
Plants take up NH
4
?
through high-affinity (HATS) and
low-affinity (LATS) transport systems, depending on the
nutrient concentration in the external medium (Wang et al.
1993). Recent studies have characterised the high-affinity
ammonium transporters (AMT1) because they act at low
soil solution N concentrations and may be involved in
NH
4
?
uptake efficiency in plants (Ranathunge et al. 2014;
Lima et al. 2010; Gu et al. 2013).
Electronic supplementary material The online version of this
article (doi:10.1007/s11816-015-0359-2) contains supplementary
material, which is available to authorized users.
&Leandro Martins Ferreira
leandromartins@ufrrj.br
1
Departamento de Solos, Universidade Federal Rural do Rio
de Janeiro, BR 465, km 7, Serope
´dica, RJ 23897-000, Brazil
2
Departamento de Quı
´mica, Universidade Federal Rural do
Rio de Janeiro, BR 465, km 7, Serope
´dica, RJ 23897-000,
Brazil
3
Centro de Biocie
ˆncias e Biotecnologia, Universidade
Estadual do Norte Fluminense Darcy Ribeiro, Av. Alberto
Lamego, 2000, Campos dos Goytacazes, RJ 28013602, Brazil
123
Plant Biotechnol Rep Online ISSN 1863-5474
DOI 10.1007/s11816-015-0359-2 Print ISSN 1863-5466
Genome sequencing identified several ammonium trans-
porters belonging to the AMT1 family in various species, such
as Oryza sativa (OsAMT1.1–1.3) (Suenaga et al. 2003;Son-
oda et al. 2003a,b), Lycopersicon esculentum (LeAMT1.1–
1.3)(vonWire
´netal.2000), Arabidopsis thaliana
(AtAMT1.1–1.5,AtAMT2) (Kaiser et al. 2002;Sohlenkamp
et al. 2002), and Triticum aestivum (TaAMT1.1–1.3)(Jahn
et al. 2004)profiles.OsAMT1.1 is an NH
4
?
-responsive gene,
expressed in the roots and shoots (Ranathunge et al. 2014),
whereas the OsAMT1.2 and OsAMT1.3 genes are specifically
expressed in the roots (Sonoda et al. 2003a,b). OsAMT1.2
responds positively to resupply with increasing doses of
ammonium, whereas OsAMT1.3 shows increased expression
at N concentrations below 0.15 mM and is repressed at higher
concentrations (Gaur et al. 2012).
The OsAMT1.2 and OsAMT1.3 gene expression profiles
in the roots suggest that both are key components of the
uptake system. However, they play different roles in N
utilisation. OsAMT1.3 appears to act as an N sensor,
whereas OsAMT1.2 acts as an NH
4
?
transporter (assimi-
lator) at low concentrations. The observation that
OsAMT1.3 was repressed not only in the presence of NH
4
?
but also in the presence of NO
3
-
has led to the hypothesis
that it may serve as an N sensor (Yao et al. 2008).
Sonoda et al. (2003a,b) suggested that NH
4
?
transporters
are regulated by the glutamine concentrations inside the roots
and not by the NH
4
?
concentrations in the external solution
(i.e., the control over the NH
4
?
uptake is internal). However,
the regulation of the AMT1 family of transporters varies with
the genotype and NH
4
?
availability (Gaur et al. 2012).
Plant varieties with increased N uptake efficiency in
soils containing low N concentrations must be selected for
sustainable agriculture (Glass et al. 2002). Therefore, the
molecular and physiological responses of plants under low
N concentrations should be well understood. Some
researchers have suggested that the OsAMT1.3 transporter
may function to signal the presence of N in the soil, in
addition to its potential involvement as a transporter in the
uptake of reduced NH
4
?
levels in the soil (Sonoda et al.
2003a,b; Gaur et al. 2012).
The present study aimed to evaluate the contribution of
the OsAMT1.3 transporter in increasing the NH
4
?
uptake
efficiency and to characterise its role in the mechanisms by
which plants adapt to environments with low N
availability.
Materials and methods
Plant material and growth conditions
Transgenic rice plants of the variety Nipponbare (Oryza
sativa L. subsp. Japonica) were used in this study. The
experiments were performed in a climatic chamber
(light/dark cycle, 12/12 h; 28/26 °C; light intensity,
500 lmol m
-2
s
-1
; relative humidity, 70 %). The rice
seeds were surface-sterilised with sodium hypochlorite
(2 %) for 10 min and germinated in distilled water. Five
days after germination (DAG), plants were transferred to
Hoagland solution (Hoagland and Arnon 1950) with dif-
ferent N regimes. The solutions were changed every
3 days, and the pH was maintained at 5.8.
Gene constructs
Transgenic rice lines were generated by expressing
OsAMT1.3 under the control of the maize ubiquitin 1
promoter (UBIL:OsAMT1.3:3xHA) using the MultiSite
Gateway cloning kit (Life Technologies, Carlsbad, CA,
USA). The amplified fragment of OsAMT1.3 was cloned
into the molecular cloning site of the pH7m34GW vector,
with the recombination sites necessary for cloning the
Gateway vectors (Table S1). To generate the green fluo-
rescent protein (GFP) and b-glucuronidase (GUS)
OsAMT1.3 construct, a 1500-bp fragment upstream of the
translation initiation site was amplified from genomic DNA
in two subsequent PCRs with hybrid primers (Table S2)
and cloned into the molecular cloning site of the pGWFS7
vector (Karimi et al. 2002) to drive GFP expression and
GUS activity. The resulting constructs were transformed
into Escherichia coli.
Genetic transformation and in vitro development
of transgenic plants
Rice plants were transformed according to the Agrobac-
terium-mediated transformation of embryogenic calli with
the OsAMT1.3 binary construct. The subsequent regener-
ation of transgenic lines and the separation of early events
of independent stable transformations within the callus
material have been described previously (Toki et al. 2006).
Transformed calli were selected via hygromycin resistance
conferred by a UBIL promoter-driven hph gene. Several T1
transformants were generated and confirmed by hygro-
mycin resistance. The transgenic lines selected were grown
in a greenhouse, and homozygous lines of the T3 genera-
tion were selected by segregation analysis for hygromycin
resistance.
Localisation studies
Transgenic rice plants (L#4) were grown for 14 days in a
Hoagland solution with two different N treatments: a
constant N supply (2.0 mM NH
4
?
) as a control or N
deficiency. The roots were harvested, infiltrated with
staining solution containing 5-bromo-4-chloro-3-indolyl-b-
Plant Biotechnol Rep
123
D-glucuronide (X-gluc), and maintained at 37 °C for 3 h
(Jefferson et al. 1987). Root segments (15 mm) from the
tips of the roots and middle third were infiltrated with a
2.5 % glutaraldehyde solution in 0.01 M phosphate buffer.
The samples were embedded in Leica
Ò
synthetic resin
(hydroxyethyl methacrylate), according to the manufac-
turer’s instructions, and sectioned using a Leica rotary
microtome. The 2-lm sections were observed on an
Axioplan light microscope (Zeiss) equipped with AxioVi-
sion software.
OsAMT1.3 promoter activity
Transgenic rice plants (L#4) were grown in a Hoagland
solution with low N availability until 10 DAG (0.5 mM
NO
3
-
) to reduce OsAMT1.3 expression after germination
and reduce the interference of this dose in the subsequent
treatments. The plants were then subjected to three dif-
ferent N regimes over 14 days: without N (control),
2.0 mM NO
3
-
, and 2.0 mM NH
4
?
. Roots were harvested
at 0, 3, 7, and 14 days after treatment and stored at -80 °C
for subsequent use.
Root samples were ground in liquid N
2
; homogenised in
three volumes of 50 mM Tris–HCl pH 8.0 buffer con-
taining 1 mM EDTA, 1.5 % polyvinylpolypyrrolidone
(PVPP), 10 mM dithiothreitol (DTT), 30 % glycerol, and
1 mM phenylmethylsulfonyl fluoride (PMSF); and then
centrifuged at 14,0009gfor 30 min. The supernatant was
used to determine the enzyme activities. The protein con-
centration was determined according to Bradford (1976).
The GUS activity was determined spectrophotometrically
using the enzyme substrate p-nitrophenyl-b-D-glucuronide
(PNPG), according to Aich et al. (2001). The activity was
expressed as DOD
405
in mg
-1
protein h
-1
.
OsAMT1.1–1.3 NH
4
1
transporter expression
Transgenic rice plants (L#2 and L#8), which overexpressed
OsAMT1.3, and WT plants were grown in a Hoagland
solution with 0.5 mM NO
3
-
, to reduce the natural
OsAMT1.3 expression after germination. At 30 DAG, the
plants were treated with a solution containing 0.5 mM
NH
4
?
. Plants were harvested at 2 and 6 h following
treatment, and root samples were stored at -80 °C for
subsequent use.
Total RNA was extracted according to GAO et al.
(2001) in NTES buffer (0.2 M Tris–HCl pH 8.0, 25 mM
EDTA, 0.3 M NaCl, 2 % SDS). The total RNA was
quantified using a Qubit 2.0 fluorometer (Life Technolo-
gies), according to the manufacturer’s instructions. The
total RNA was treated with DNaseI (Life Technologies)
and used for cDNA synthesis using a High-Capacity RNA-
to-cDNA
TM
kit (Life Technologies) and oligo(dT) primers,
according to the manufacturer’s instructions. qRT-PCR
was performed using the Power SYBR
Ò
Green PCR Master
Mix kit and a StepOne real-time PCR system (Applied
Biosystems, Carlsbad, CA, USA). The PCR program con-
sisted of 95 °C for 15 s and 60 °C for 60 s. Two qRT-PCR
determinations were performed for each cDNA sample.
The threshold cycle (C
t
) values for each sample were
normalised with the O. sativa elongation factor (eEF1-a)as
a housekeeping gene. The relative quantity was calculated
using the 2
-DDCT
method (Livak and Schmittgen 2001).
The primers designed by Duan et al. (2007) for the
ammonium transporter genes (OsAMT1.1,1.2, and 1.3)
were used.
NH
4
1
uptake kinetics of rice lines overexpressing
the OsAMT1.3 gene
Transgenic rice plants (L#2 and L#8) were grown as pre-
viously described. At 27 DAG, the plants were submitted
to N starvation for 72 h, followed by resupply with 0.2 mM
NH
4
?
. Samples of nutrient solution (1.0 mL) were col-
lected at intervals of 30 min until N exhaustion. At the end
of the experiment, shoots and roots were harvested
(Table S3). The kinetic parameters, V
max
and K
M
, were
measured by depletion of NH
4
?
in the uptake solution over
time according to Claassen and Barber (1974). The
ammonium content of the nutrient solution was determined
according to Felker (1977). The integrated analysis of
NH
4
?
uptake was calculated as a=V
max
/K
M
(Marschner
1995). In the final samples of nutrient solution, the con-
centration at which net uptake of ions ceases before the
ions are completely depleted was measured (C
min
)
(Marschner 1995). Root parameters (root length, surface
area, projected area, volume, and number of tips) were
determined using the Winrhizo 4.1 software (Regent
Instruments, Quebec, Canada).
Statistical analysis
A completely randomised experimental design was uti-
lised, with four replicates in all experiments. Analysis of
variance was performed by applying the Ftest, the aver-
ages were compared using a Scott–Knott test at pB0.05,
and the standard error was calculated.
Results
OsAMT1.3 tissue-specific localisation
and quantification
To identify the sites of action of the OsAMT1.3 transporter
and study its possible involvement in the adaptation of
Plant Biotechnol Rep
123
plants to low N supplies, rice plants (L#4) were grown
under high NH
4
?
supply or N starvation for 14 days. The
roots were collected and infiltrated with a solution con-
taining the b-glucuronidase (X-gluc) substrate (Fig. S1).
Under treatment by 2.0 mM of NH
4
?
, no promoter activity
was observed (Fig. 1a), whereas under N starvation, an
intense blue stain was observed (Fig. 1b). Two segments of
roots (black boxes) were selected for further analyses:
15 mm from the tip and the middle third (Fig. 1c, d). In
these segments, intense GUS and GFP activities were
observed close to the epidermis, at the zones of lateral root
emission and at the tips of the lateral roots (Fig. 1e–h).
Histological sections of the selected segments are shown in
Fig. 2a–f.
Cross sections obtained from the tips and from the
middle third of the root exhibited different patterns of
staining (Fig. 2). Cross section from the tips showed
OsAMT1.3 activity in the exodermis, sclerenchyma, cortex,
and stele (Fig. 2a, b), while in cross and longitudinal sec-
tions from the middle third region, with longer lateral roots,
a higher OsAMT1.3 activity was observed in the sites of
lateral root emission and at the exodermis (Fig. 2c–f;
Fig. S2).
In addition to the histochemical assays (Figs. 1,2), the
in vitro GUS activity was also quantified under treatment
with NH
4
?
or NO
3
-
and under N deficiency for 14 days.
High GUS activity was observed in plants (L#4) grown
without N at 3 days following the treatment (Fig. 3). This
high activity remained until 7 days and then decreased
after 14 days. High OsAMT1.3 activity was observed in all
periods analysed under N deprivation. No changes were
noted in the GUS activity in plants grown under a constant
N supply. These results indicated that the OsAMT1.3
ammonium transporter is induced strongly by N deficiency
and repressed under NO
3
-
or NH
4
?
supply.
OsAMT1.3 positively affects OsAMT1.2 expression
Rice plants overexpressing OsAMT1.3 were obtained to
examine the effect of its expression on the NH
4
?
uptake
and root growth. L#2 and L#8 were selected for further
study because they showed high OsAMT1.3 expression
levels. Rice lines showed increased levels of OsAMT1.3
expression under constant N supply (Fig. 4a). In addition,
OsAMT1.2 expression showed the same pattern as
OsAMT1.3 (Fig. 4a, b; Fig. S3). The OsAMT1.2 expression
levels were higher at 2 h than at 6 h (Fig. 4c, d), and the
lines did not present significant changes in their OsAMT1.1
expression levels at 2 and 6 h (Fig. 4c).
Uptake kinetics and root parameters under low
NH
4
1
supply and N deficient
The overexpression of the OsAMT1.3 did not alter the V
max
significantly in the rice lines; however, the K
M
values were
26.4 and 52.4 % lower for L#2 and L#8, respectively, than
for the WT (Table 1). The integrate analysis of NH
4
?
uptake using the avalue indicated that the rice lines
showed kinetic parameters more favourable to the uptake
of NH
4
?
at low concentrations than the WT. Furthermore,
Fig. 1 Rice roots (L#4) grown with 2.0 mM NH
4
?
(a) and under N
starvation (scale bar 10 mm) (b). Black boxes indicate the root
segments selected for the anatomical sections. Middle third (c) and tip
(d)(scale bar 2.0 mm). An intense blue stain (GUS) and fluorescence
(GFP) were observed in the lateral root emission zone (scale bar
100 lm) (e,f) and in the tips (scale bar 400 lm) (g,h)
Plant Biotechnol Rep
123
a significant reduction in C
min
values of 4.2 and 17 % was
observed for L#2 and L#8, respectively (Table 1). When
plants were grown in N-deficient medium or 0.2 mM NH
4
?
for 14 days, several root parameters were modified in the
rice lines. Greater root length, projection area, surface area,
root volume, and number of tips were observed for L#2 and
L#8 in both treatments (Table 2).
Despite the higher values observed for the root param-
eters in 0.2 mM NH
4
?
, the differences between the lines
and WT were more evident without N. L#2 and L#8
showed greater increases in the root length, approximately
32 and 48 %, respectively, without N, while with 0.2 mM
NH
4
?
, the increases were 12 and 20 % for L#2 and L#8,
respectively. In addition, marked increases in the number
of tips were observed for L#2 and L#8 (44 and 77 %,
respectively) during N starvation, while under the treat-
ment with 0.2 mM NH
4
?
a minor increase was observed: 4
and 7 %, for L#2 and L#8, respectively.
Fig. 2 Anatomical sections of
rice roots (L#4) after 14 days
without N. Blue staining (GUS)
was observed at the segment of
tips (a,b)(scale bar 100 lm)
and in the middle third with
lateral roots (c–f)(scale bar
50 lm). Arrows indicate the
OsAMT1.3 promoter activity
zones. ccortex, eexodermis
Root
Time (days)
03714
β - glucuronidase activity
(Δ DO405 mg-1 protein. h-1)
0.0
0.1
0.2
0.3
0.4
N deprivation
2.0 mM NO3-
2.0 mM NH4+
Fig. 3 OsAMT1.3 activity in rice roots (L#4) under N starvation,
2.0 mM NO
3
-
or 2.0 mM NH
4
?
supply. Roots were harvested at 0, 3,
7, and 14 days after treatment. Values represent averages ±SE
(n=4)
Plant Biotechnol Rep
123
Discussion
In this study, transgenic rice plants (L#4) were submitted to
N deprivation or 2.0 mM NH
4
?
for 14 days to identify
whether the promoter activity is regulated by N and the
sites of action of OsAMT1.3. The OsAMT1.3 activity was
restricted to the roots under N deficiency (Fig. 1), sup-
porting the data found by Sonoda et al. (2003a,b) and Yao
et al. (2008). The plants subjected to N deprivation showed
higher OsAMT1.3 promoter activity, mainly in the root
emission zone and roots tips (Fig. 1e–h). Furthermore, root
sections at the segment from the tips and middle third
exhibited different patterns of OsAMT1.3 promoter activity
(Fig. 2). In the tips, the OsAMT1.3 activity was uniformly
distributed at the exodermis, sclerenchyma, cortex, and
stele (Fig. 2a, b), while in the middle third segment, it was
observed only at the cortex and exodermis (Fig. 2c–f). The
ammonium taken up by the AMTs is readily assimilated by
cytosolic GS1 and NADH-GOGAT isozymes in the surface
cell layers of the roots, epidermis, and exodermis (Hirose
et al. 1997; Ishiyama et al. 1998). The OsAMT1.3 promoter
activity observed mainly in the exodermis (Fig. 2c, d) is
related to the sites of primary ammonium assimilation,
indicating a role of the encoded protein in the ammonium
uptake. The OsAMT1.3 promoter activity at root emission
zones also suggested a role in signalling events that result
in lateral root emission under N deficiency (Fig. 2e, f;
Fig. S2). Yao et al. (2008) observed that OsAMT1.3 was
expressed preferentially at the apex of the lateral and
seminal roots. These data support the idea that OsAMT1.3
acts as a sensor for nutrients present in the soil, changing
the plant metabolism through the activation of signal
transduction pathways (Gojon et al. 2011).
In A. thaliana with a quadruple knockout of ammonium
transporter genes (amt1.1,amt1.2,amt1.3, and amt2.1), no
lateral root formation was observed, and the lateral root
formation decreased significantly in a atamt1.3 mutant
under conditions of low N, supplied as NH
4
?
and NO
3
-
(Lima et al. 2010). These authors suggested that AtAMT1.3
might be involved in triggering lateral root formation in
Arabidopsis. The AtAMT1.3 ammonium transporter does
not show high sequence similarity with OsAMT1.3 (Li
et al. 2009); however, OsAMT1.3 could also act as a signal
for the emission of lateral roots under N deficiency in rice
(Fig. 2c–f).
The OsAMT1.3 activity in plants grown in nutrient
solution without N was higher at 3 and 7 days after treat-
ment. When these plants were transferred to solutions
containing N as NH
4
?
or NO
3
-
ions, no changes were
observed in the OsAMT1.3 promoter activity (Fig. 3). This
result is consistent with the data of Sonoda et al. (2003a,b),
where rice plants submitted to N starvation showed
upregulation of OsAMT1.3 expression, while in plants
submitted to N supply as NH
4
?
or NO
3
-
, the opposite
AMT1.3
a
Relative Expression (2
-ΔΔCT
)
0
2
4
6
8
10
12
14
WT
L#2
L#8
AMT1.1
c
2 h 6 h
Relative Expression (2
-ΔΔCT
)
0
1
2
3
4
AMT1.2
b
Relative Expression (2
-ΔΔCT
)
0
2
4
6
8
10
12
14
Fig. 4 Relative expression of three ammonium transporters
(OsAMT1.3,OsAMT1.2, and OsAMT1.1) in the roots of wild type
(WT), L#2, and L#8 rice under a constant 0.5 mM NO
3
-
/NH
4
?
supply (a–c). WT plants at 2 h were used as the reference. Values are
averages ±SE (n=3)
Table 1 NH
4
?
uptake kinetic
parameters (V
max
,K
M
,a, and
C
min
) for wild type (WT), L#2,
and L#8 plants under resupply
with 0.2 mM NH
4
?
Lines V
max
(lmol g
-1
h
-1
)K
M
(lmol L
-1
)a(V
max
/K
M
)(Lg
-1
h
-1
)C
min
(lmol L
-1
)
WT 9.28a 24.52a 0.38c 26.24a
L#2 7.68b 18.04b 0.48b 25.13a
L#8 9.39a 11.68c 0.82a 21.76b
Averages followed by the same letter within the same column do not differ significantly according to the
Scott–Knott test at pB0.05
Plant Biotechnol Rep
123
behaviour was observed: downregulation of OsAMT1.3
expression. Gaur et al. (2012) reported that the repression
of OsAMT1.3 through an increase in N might not be a
universal mechanism, but may depend on the genotype and
on the N level required by a given genotype. These authors
observed repression of OsAMT1.3 with increasing NH
4
?
concentrations in the solution, up to 1.0 mM, for the rice
cultivar Kalanamak 3119, whereas the cultivar Pusa Bas-
mati showed the opposite behaviour, increasing the
OsAMT1.3 expression under higher NH
4
?
levels. The
Nipponbare rice variety used in our study requires low N
supply and also showed high OsAMT1.3 expression under
N deficiency (Fig. 3). OsAMT1.3 expression is believed to
be useful as a biomarker to determine the optimal N supply
for rice varieties adapted to low and high N environments.
These differences in induction of the high-affinity AMT
genes might be attributed to differences in N perception
and signalling (Gaur et al. 2012).
Thus, overexpression of members of the AMT1 family
might be useful to improve N uptake from soils with low
NH
4
?
concentrations (Ranathunge et al. 2014). However,
some members of the AMT1 family may not be directly
involved in the acquisition of NH
4
?
from the external
solution, acting instead as sensors of the intracellular NH
4
?
status (Hoque et al. 2006). Thus, we developed rice plants
overexpressing OsAMT1.3 to identify whether the gene
product is involved in ammonium uptake or signalling.
The relative expression of the high-affinity ammonium
transporter genes (OsAMT1.1–1.3) was performed at 2 and
6 h with a constant N supply (Fig. 4). L#2 and L#8 showed
high OsAMT1.3 expression (Fig. 4a). In addition, a strong,
positive correlation was observed between the OsAMT1.3
and OsAMT1.2 expressions at 2 and 6 h (Fig. 4a, b; Fig. S3).
No significant change was observed in the expression of the
OsAMT1.1. These data support the hypothesis that the
overexpression of OsAMT1.3 could alter the expression of
other members involved in high-affinity ammonium trans-
port, such as OsAMT1.2. At 6 h, a decrease in the expression
of OsAMT1.2 was observed, possibly indicating negative
feedback regulation by glutamine (Sonoda et al. 2003a,b).
Rice lines overexpressing OsAMT1.3 showed lower K
M
and C
min
values than WT plants when supplied with
0.2 mM NH
4
?
(Table 1). This result suggested that
OsAMT1.3 overexpression, associated with a higher
OsAMT1.2 expression (Fig. 4), resulted in increased
uptake efficiency by these plants, considering that
OsAMT1.2 is involved in NH
4
?
uptake from soil solutions
(at concentrations\200nM)andintheretrievalofNH
4
?
in the vascular system (Sonoda et al. 2003a,b). The
AMT1 family might show different K
M
values at low
concentrations of NH
4
?
,asobservedinArabidopsisand
maize plants (Gazzarrini et al. 1999 andGuetal.2013).
This suggested that the higher expression of OsAMT1.3
and OsAMT1.2 changed the K
M
of the NH
4
?
high-affinity
transport system, resulting in increased uptake efficiency.
The combination of low K
M
and C
min
values associated
with increased root growth is a desirable characteristic in
crop plants because it equates to increased N uptake effi-
ciency (Barber 1995). In addition to improved kinetic
parameters, the rice lines showed longer roots with more
tips, indicating that OsAMT1.3 expression might contribute
to an increase in lateral root emission under N deficiency or
low N supply (Table 2). A greater difference was observed
for root length and number of tips during N starvation,
indicating a contribution of the natural OsAMT1.3
expression to the root parameters.
Our results showed that overexpression of OsAMT1.3
was associated with the natural expression of OsAMT1.2,
and promoted the uptake of NH
4
?
at low concentrations
and changes to the root morphology of the lines. However,
further studies should be carried out to isolate the role of
the OsAMT1.3 using knockout plants.
Acknowledgments This study was supported by the National
Council for Scientific and Technological Development (Conselho
Nacional de Desenvolvimento Cientı
´fico e Tecnolo
´gico-CNPq), the
Research Support Foundation of the State of Rio de Janeiro (Funda-
c¸a
˜o de Amparo a
`Pesquisa do Estado do Rio de Janeiro-FAPERJ), and
the Coordination for the Improvement of Higher Education Personnel
(Coordenac¸a
˜o de Aperfeic¸ oamento de Pessoal de Nı
´vel Superior-
CAPES).
Table 2 Root parameters for wild type (WT), L#2, and L#8 plants grown in a nutrient solution without N or with 0.2 mM NH
4
?
for 14 days
Rice plants Treat. Length (cm) Proj. area (cm
2
) Surf. area (cm
2
) Root vol. (cm
3
) Tips
WT Without N 366.2b 99.7b 313.3b 19.8b 184.1b
L#2 485.2a 136.5a 411.4a 27.8a 265.9a
L#8 543.9a 133.4a 419.2a 25.9a 327.0a
WT 0.2 mM NH
4
?
614.2b 154.5b 499.5b 35.8b 432.3b
L#2 691.3a 205.8a 648.7a 48.8a 450.2a
L#8 739.7a 206.5a 667.8a 45.5a 466.6a
Treat. Treatment, proj. area projected area, surf. area surface area, root vol. root volume
Averages followed by the same letter within the same column do not differ significantly according to the Scott–Knott test at pB0.05
Plant Biotechnol Rep
123
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