Annals of Botany 99: 1153–1160, 2007
doi:10.1093/aob/mcm051, available online at www.aob.oxfordjournals.org
Responses of Rice Cultivars with Different Nitrogen Use Efficiency to Partial
Nitrate Nutrition
Y. H . D U AN , Y. L . Z H AN G, L . T. Y E , X . R . FAN , G . H . XU and Q . R . S HE N*
College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, 210095, PR China
Received: 24 November 2006 Returned for revision: 11 December 2006 Accepted: 8 February 2007 Published electronically: 11 April 2007
† Background and Aims There is increased evidence that partial nitrate (NO2
3 ) nutrition (PNN) improves growth of
2
rice (Oryza sativa), although the crop prefers ammonium (NHþ
4 ) to NO3 nutrition. It is not known whether the
response to NO2
3 supply is related to nitrogen (N) use efficiency (NUE) in rice cultivars.
† Methods Solution culture experiments were carried out to study the response of two rice cultivars, Nanguang
(High-NUE) and Elio (Low-NUE), to partial NO2
3 supply in terms of dry weight, N accumulation, grain yield,
NHþ
4 uptake and ammonium transporter expression [real-time polymerase chain reaction (PCR)].
2
† Key Results A ratio of 75/25 NHþ
4 -N/NO3 -N increased dry weight, N accumulation and grain yield of ‘Nanguang’
by 30, 36 and 21 %, respectively, but no effect was found in ‘Elio’ when compared with those of 100/0 NHþ
4 -N/
15
2
þ
NO2
N-NHþ
3 -N. Uptake experiments with
4 showed that NO3 increased NH4 uptake efficiency in ‘Nanguang’
þ
by increasing Vmax (14 %), but there was no effect on Km. This indicated that partial replacement of NH4 by
NO2
3 could increase the number of the ammonium transporters but did not affect the affinity of the transporters
for NHþ
4 . Real-time PCR showed that expression of OsAMT1s in ‘Nanguang’ was improved by PNN, while that
in ‘Elio’ did not change, which is in accordance with the differing responses of these two cultivars to PNN.
† Conclusions Increased NUE by PNN can be attributed to improved N uptake. The rice cultivar with a higher NUE
has a more positive response to PNN than that with a low NUE, suggesting that there might be a relationship
between PNN and NUE.
þ
Key words: Ammonium transporter, partial NO2
3 nutrition, NH4 uptake, nitrogen use efficiency, rice, Oryza sativa.
IN TROD UCT IO N
Nitrogen (N) is one of the essential macronutrients for rice
(Oryza sativa L.) growth and one of the main factors to be
considered for developing a high-yielding rice cultivar. In a
2
paddy field, ammonium (NHþ
4 ) rather than nitrate (NO3 )
tends to be considered the main source of N for rice
(Wang et al., 1993). However, in recent years, researchers
have paid more and more attention to the partial NO2
3 nutrition (PNN) of rice crops, and their results have shown that
lowland rice was exceptionally efficient in absorbing NO2
3
formed by nitrification in the rhizosphere (Kirk and
Kronzucker, 2005; Duan et al., 2006).
Rice roots can aerate the rhizosphere by excreting oxygen
(O2). Kirk (2001) reported that substantial quantities of
NO2
3 were produced in the rhizosphere of rice plants
through nitrification, and microbial nitrification was partially responsible for the maximum overall rate of microbial
O2 consumption. Most recently, using model calculations
and experiments, Kirk and Kronzucker (2005) and
Kronzucker et al. (1999, 2000) concluded that NO2
3
uptake by lowland rice might be far more important than
was previously thought; its uptake rate could be comparable
with that of NHþ
4 , and it could amount to one-third of the
total N absorbed by rice plants. Therefore, although the predominant species of mineral N in bulk soil for paddy rice
fields is likely to be NHþ
4 , rice roots are actually exposed
to a mixed N supply in the rhizosphere (Briones et al.,
2003; Y. L. Li et al., 2006).
* For correspondence. E-mail [email protected]
When rice plants in solution culture were fed with a
2
mixture of NHþ
4 and NO3 compared with either of the N
sources applied alone at the same concentration, yield
increases of 40– 70% were observed (Heberer and Below,
1989; Qian et al., 2003). The growth and N acquisition
of rice were significantly improved by the addition of
þ
NO2
3 to nutrition solution with NH4 alone (Cox and
Reisenauer, 1973; Raman et al., 1995; Duan et al., 2006).
The increased N acquisition could be attributed to the
2
increased influx of NHþ
4 by NO3 (Kronzucker et al.
þ
1999); NH4 is taken up by plant roots through ammonium
transporters (AMTs).
The first AMT was isolated from Arabidopsis
(Ninnemann et al., 1994). Later, AMTs were isolated
from Brassica napus (Pearson et al., 2002), Lycopersicon
esculentum (Lauter et al., 1996; von Wiren et al., 2000),
Nicotiana tabacum ‘Samsun’ (Matt et al., 2001) and
Lotus japonicus (Salvemini et al., 2001; Simon-Rosin
et al., 2003). AMTs in rice roots were first identified by
Suenaga et al. (2003) and they could be classified into
two types: high-affinity transport system (HAT) and lowaffinity transport system (LAT) (Howitt and Udvardi,
2000; Loque and von Wiren, 2004). At low NHþ
4 concentration, uptake is mediated by HATs and exhibits sensitivity
to metabolic inhibitors (Wang et al., 1993). At high NHþ
4
concentration (between 1 and 40 mM), uptake is mediated
by LATs and is less responsive to metabolic inhibitors
(Wang et al., 1994). There are four AMT families in rice,
i.e. OsAMT1, OsAMT2, OsAMT3 and OsAMT4, based on
their phylogenic relationships (Suenaga et al., 2003). The
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1154
Duan et al. — Responses of Rice to Partial Nitrate Nutrition
OsAMT1s (OsAMT1;1, OsAMT1;2 and OsAMT1;3) share
high sequence similarity to each other and are very dissimilar to the other three OsAMT families (Sonoda et al.,
2003). The expression pattern of HAT-OsAMT1s
(OsAMT1;1 – 1;3) was distinct and regulated at least in
part by the N source, such as NHþ
4 and N starvation
(Suenaga et al., 2003; B. Z. Li et al., 2006). In contrast,
the expression of OsAMT1s in response to NO2
3 in different
rice cultivars is still unknown and should be studied further.
Nitrogen use efficiency (NUE), defined as the ratio of
grain yield to supplied N, is a key parameter for evaluating
a crop cultivar, and it is composed of N uptake efficiency
and N physiological use efficiency (De Macale and Velk,
2004). Nitrogen uptake efficiency is the N accumulation
relative to its supply, while N physiological use efficiency
represents grain yield relative to N accumulation (Moll
et al., 1982). While the amount of N available from soil
and fertilizer is difficult to measure, grain yields can be
used for evaluating the NUE, and high-NUE cultivars can
be defined by their ability to produced higher grain yields
than others under the same experimental conditions
(Ladha et al., 1998). As PNN in rice could improve the
growth and increase grain yield, theoretically it should
increase NUE, but this relationship still has to be verified.
In this study, the growth and NHþ
4 uptake of two rice
cultivars with differing NUEs were reinvestigated, and
then the expression of OsAMT1s (OsAMT1;1– 1;3) under
2
NHþ
4 nutrition with and without NO3 was characterized.
Finally a possible relationship between PNN and NUE
was proposed
(early tillering stage), 60 d (maximum tillering stage),
90 d (heading stage) and 150 d (mature stage).
The whole growth period experiment
Seven-day-old seedlings with uniform size and vigour
were transplanted into holes in a lid placed over the top
of pots (20 holes in a lid and two seedlings per hole). All
pots were filled with 5 L of Yoshida nutrient solution
(Yoshida et al., 1972). After the maximum tillering stage,
all plants were transferred to pots containing 20 L of nutrient solution with three rice seedlings per pot (three holes in
a lid and one seedling per hole). The rice seedlings were
2
subjected to two treatments of different NHþ
4 -N/NO3 -N
ratios, i.e. 100/0 and 75/25, by adding 2.86 mM N in the
form of either (NH4)2SO4 or a mixture of (NH4)2SO4 and
NH4NO3. The nutrient solution contained the following
macronutrients in mM: NaH2PO4, 0.3; K2SO4, 2.0; CaCl2,
1.0; MgSO4, 1.5; Na2SiO3, 1.7, and the following micronutrients in mM: Fe-EDTA, 20; MnCl2, 9.1; (NH4)6Mo7O24,
0.4; H3BO3, 37; ZnSO4, 0.8; CuSO4, 0.3. To inhibit nitrification, 7 mM dicyandiamide (DCD-C2H4N4) was mixed into all
the solutions. The nutrient solution was renewed every
þ
2
3 d. No NO2
3 was detected in the 100/0 NH4 -N/NO3 -N treatment. The pH of all the nutrient solutions was adjusted daily
to 5.5 with 0.1 M NaOH or 0.1 M HCl.
At each harvest, rice roots and shoots were separated and
washed, then placed in an oven at 105 8C for half an hour to
inactivate the enzymes, and finally dried to a constant
weight at 70 8C. The dry weight was recorded. Nitrogen
content in plants was determined by the Kjeldahl method
(Chu et al., 2004).
M AT E R I A L S A N D M E T H O D S
Plant materials
15
N-labelled growth experiment
Two Japonica rice (O. sativa L.) cultivars ‘Nanguang’ and
‘Elio’ were chosen based on their different responses to N
application in the field trials of 187 Japonica rice cultivars
carried out in 2003 and 2004 (Zhang et al., 2007). Their
agronomic traits are shown in Table 1. ‘Nanguang’ had a
high grain yield under low N treatment and responded
well to increasing N supply, and was thus identified as a
high-NUE cultivar. ‘Elio’ produced a lower grain yield
and thus was defined as a low-NUE cultivar.
All the hydroponic experiments in this study were carried
out in the greenhouse with temperatures ranging from 20 8C
at midnight to 35 8C at mid-day during the period 12 April
2004 to 1 October 1 2004 at Nanjing Agricultural
University, China. After germination, rice plants were
grown in nutrient solution for 30 d (seedling stage), 45 d
Rice plants were cultivated as described above and
treated with different N forms (100/0 and 75/25 of
15
2
þ
NHþ
N [(NH4)2SO4,
4 -N/NO3 -N). NH4 was labelled by
15
.
10 7 % atom N excess] in the treatments.
Plant samples were collected at the seedling, early and
maximum tillering stages. Nitrogen content in the dried
samples was determined by the Kjeldahl method, and the
15
N abundance in each fraction was determined using a
MAT251 isotope mass spectrometer.
Kinetics of
15
N-labelled NHþ
4 uptake
Rice seedlings with four leaves (30 d after germination)
were prepared in a nutrient solution containing 1.43 mM
TA B L E 1. Characteristics of ‘Nanguang’ and ‘Elio’ rice cultivars evaluated in field experiments (N ¼ 180 kg ha21) in 2004
(Zhang et al., 2007)
Cultivars
‘Nanguang’
‘Elio’
Nitrogen use efficiency
(kg kg21)
Grain yield
(t ha21)
Total biomass weight
(t ha21)
Growth duration
(d)
Tillers/
plant
Plant height
(cm)
1000-grain weight
(g)
37
30
9.12
7.83
18.7
13.8
163
157
7.3
3.4
108
96
26.8
39.7
Duan et al. — Responses of Rice to Partial Nitrate Nutrition
NH4NO3 as N source; they were starved of N in a solution
with no N for 2 d. Then, they were placed in a series of
nutrient solutions containing 15NHþ
4 in the form of
(15NH4)2SO4 at concentrations of 0.025, 0.05, 0.1, 0.15,
0.2, 0.3, 0.4, 0.8 and 1.2 mM. To study the effect of NO2
3
.
on 15NHþ
4 uptake kinetics, 0 25 mM Ca(NO3)2 was added
to the series of 15NHþ
4 -containing solutions. The concentrations of other nutrients were not changed.
At 09:00 h, roots of three identical rice seedlings were
immersed in a black cloth-wrapped glass test tube containing 20 mL of 15NHþ
4 -containing solutions for 2 h at 30 +
1 8C and a light intensity of 900 mmol photos m22 s21 in
a growth chamber. Each tube was weighed at the beginning
and at the end of the experiment to calculate the water loss
through evaporation and transpiration during the period.
Each treatment had three replicates. The NHþ
4 concentration
of the external solution and the uptake rate were fitted to
the Michaelis – Menten equation to obtain the kinetic parameters of Vmax and Km (Eisenthal and Cornish-Bowden,
1974).
RNA extraction and real-time PCR
After cultivation in a nutrient solution with 1.0 mM
(NH4)2SO4 as N source for 30 d after germination, rice
seedlings were transferred to a nutrient solution with no
N for 7 d. Then, half of the seedlings were transferred
þ
2
to a partially replaced NHþ
4 solution (75/25 NH4 /NO3 )
and the other half to an NHþ
-only
solution
with
the
4
same total N concentration at 2 mM. Two hours later,
the root tips (1 – 2 cm) and middle sections (4 cm) of
the first two fully expanded leaves were excised, immediately frozen in liquid nitrogen and stored at –80 8C until
analysis.
Total RNA from 100 mg of plant material was isolated
using Trizol reagent (Invitrogen, Carlsbad, CA, USA).
Approximately 2 mg of total RNA from each sample was
used as template for the first-strand cDNA synthesis,
which was performed using M-MLV reverse transcriptase
(Promega Madison, WI, USA) in a reaction volume of
25 ml containing 1 PCR buffer, 1 mM dNTPs, 0.5 mM
oligo(dT) primer (Promega) and 0.5 U of RNase inhibitor
(TaKaRa). The PCR amplification was performed using
Takara Ex-TaqTM polymerase for target genes and actin.
For polymerase chain reaction (PCR), the primers
(Table 2) for OsAMT1;1 – 1;3 amplification were designed
1155
according to sequences in the NCBI database (http://www.
ncbi.nlm.nih.gov/). Actin (OsRac1) was used as internal
standard in real-time PCR experiments, and the relative
expression of target genes was calculated as copies of
gene/copies of Actin.
Amplification of real-time PCR products was carried out
with a single Color Real-Time PCR Detection System
(MyiQTM Optical Module, Bio-Rad, Hercules, CA, USA)
in a reaction mixture of 20 mL containing: 0.5 mL of each
primer (10 pmol L21) for target genes or Actin (Table 2),
10 mL of SYBR Green PCR master mix [TaKaRa
Biotechnology (Dalina) Co., Ltd], 2 mL of cDNA and
7 mL of RNase-free water. The real-time PCR conditions
were as follows: denaturation at 95 8C for 30 s; followed
by 40 cycles at 95 8C for 10 s, 55 8C for 20 s, and 72 8C
for 30 s; followed by 95 8C for 1 min and 55 8C for
1 min; and followed by 80 cycles to obtain a melting
curve. Each quantification target was amplified in triplicate
samples. The target gene and actin standards in 1, 1 : 10,
1 : 100 and 1 : 1000 dilutions were always present in the
experiments (Tsuchiya et al., 2004; Yuko et al., 2004;
Jain et al., 2006).
Calculations and data analysis
The natural 15N abundance in rice without feeding 15N
was determined as background. Labelled N (15N) content
was calculated according to Sheehy et al. (2004).
15
N uptake ¼ ½ð15 NðLþSÞ %WðLþSÞ TNðLþSÞ %Þ
þ ð15 NR %WR TNR %Þ
15
N-NHþ
4 uptake efficiencyð%Þ
¼ 15 N uptakeðmgÞ=15 N supplyðmgÞ 100
where W(Lþ S) is the weight of leaves and stems in each pot,
WR is the weight of roots in each pot, TN% is the total nitrogen percentage in the plant, 15N% is the 15N atom% excess
(15N atom% excess ¼ 15N atom% excess in a labelled
plant – 15N atom% excess, in an unlabelled plant).
Statistical analyses were conducted using SPSS software
(SPSS 11.0.0, SPSS Inc., 2001) and Sigmaplot System (sigmaplot 2000, 1986– 2000 SPSS Inc.).
TA B L E 2. Primers for OsAMT1;1, OsAMT1;2, OsAMT1;3 and Actin genes
Target genes
GenBank accession no.
OsAMT1;1
AF289477
OsAMT1;2
AF289478
OsAMT1;3
AF289479
Actin
NM_197297
Direction
Sequence of primers
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
50 -GGTCATCTTCGGGTGGGTCA-30
50 -CGTGCCGTGTCAGGTCCAT-30
50 -GAAGCACATGCCGCAGACA-30
50 -GACGCCCGACTTGAACAGC-30
50 -GCGAACGCGACGGACTA-30
50 -GACCTGTGGGACCTGCTTG-30
50 -TTATGGTTGGGATGGGACA-30
50 -AGCACGGCTTGAATAGCG-30
1156
Duan et al. — Responses of Rice to Partial Nitrate Nutrition
R E S U LT S
Dry weight and N accumulation
PNN led to a significant increase of dry matter production
in ‘Nanguang’, a high-NUE rice cultivar, but no difference
was observed in ‘Elio’, a low-NUE rice cultivar, as compared with NHþ
4 only (Fig. 1). Nitrogen accumulation in
‘Nanguang’ was also increased by PNN, while no difference in ‘Elio’ was found (Fig. 2). Moreover, these effects
were more apparent in the earlier than in the later stages
(Figs 1 and 2), suggesting that partial replacement of
2
NHþ
4 by NO3 is more effective in early growth stages of
rice plants.
Grain yield and N physiological use efficiency
Grain yield of ‘Nanguang’ was higher than that of ‘Elio’
under NHþ
4 -only cultivation (Table 3). PNN led to a 21 %
increase in grain yield in ‘Nanguang’ while there was no
effect in ‘Elio’.
Nitrogen physiological use efficiencies of ‘Nanguang’
and ‘Elio’ were constant with or without NO2
3 (Table 3),
though the yield and N accumulation of ‘Nanguang’ were
improved by PNN.
NHþ
4
15
accumulation and uptake efficiency at early
growth stages
PNN increased 15NHþ
4 accumulation of ‘Nanguang’ at
the seedling stage and maximal tillering stage by 13 and
10 % in the leaves, and by 23 and 27 %, respectively, in
the roots as compared with those in the NHþ
4 -only treatment
þ
2
þ
F I G . 2. Effect of partial NO2
3 nutrition (100/0 NH4 /NO3 and 75/25 NH4 /
21
2
NO3 ) on the N accumulation (mg pot ) of ‘Nanguang’ and ‘Elio’ rice cultivars at four growth stages: seedling stage (S); maximum tillering stage (T);
heading stage (H); and maturity stage (M). Each value was the average of
three replicates. Lower case letters show the statistical significance
2
þ
2
(P , 0.05) of the treatments (100/0 NHþ
4 /NO3 and 75/25 NH4 /NO3 ) for
a given growth stage in ‘Nanguang’ or ‘Elio’ cultivars.
(Table 4). It was less effective in ‘Elio’ except at the early
tillering stage.
15
NHþ
4 uptake efficiency of ‘Nanguang’ was increased by
PNN at all three growth stages, while no difference was
observed in ‘Elio’ (Table 5). The increase could be as
high as 51 % in leaves at the seedling stage, and 70 % in
roots at the maximal tillering stage.
Kinetic parameters of
NHþ
4 net uptake
15
.
PNN increased the uptake rate (Vmax) of 15NHþ
4 by 14 1 %
in ‘Nanguang’, while there was no change in ‘Elio’ (Table 6),
indicating that the number of transporters for NHþ
4 uptake in
‘Nanguang’ is significantly increased. However, Km values
for both cultivars showed no significant difference,
suggesting that PNN does not affect the affinity of the transporters for NHþ
4 in rice roots.
þ
2
TA B L E 3. Effect of partial NO2
3 nutrition (100/0 NH4 /NO3
þ
2
and 75/25 NH4 /NO3 ) on grain yield and physiological N use
efficiency of ‘Nanguang’ and ‘Elio’ rice cultivars in a
hydroponic culture system
Cultivars
‘Nanguang’
NO2
3
2
NHþ
4 /NO3
NHþ
4/
F I G . 1. Effect of partial
nutrition (100/0
and 75/25
21
NO2
3 ) on the dry weight (g pot ) of ‘Nanguang’ and ‘Elio’ rice cultivars
at four growth stages: seedling stage (S); maximum tillering stage (T);
heading stage (H); and maturity stage (M). Each value was the average
of three replicates. Lower case letters show the statistical significance
2
þ
2
(P , 0.05) of the treatments (100/0 NHþ
4 /NO3 and 75/25 NH4 /NO3 )
for a given growth stage in ‘Nanguang’ or ‘Elio’ cultivars.
‘Elio’
2
NHþ
4 /NO3
Grain yield
(g pot21)
Physiological N use
efficiency (%)
100/0
75/25
100/0
75/25
14.7 + 1.07a
17.8 + 0.82b
9.80 + 0.76a
10.5 + 0.88a
18.5 + 0.69a
18.8 + 0.56a
12.6 + 1.02a
13.3 + 0.87a
Each value was the average of three replicates. Superscript letters show
2
the statistical significance (P , 0.05) of the treatments (100/0 NHþ
4 /NO3
2
and 75/25 NHþ
/NO
)
in
‘Nanguang’
or
‘Elio’
cultivars.
4
3
Duan et al. — Responses of Rice to Partial Nitrate Nutrition
1157
þ
2
þ
2
15
TA B L E 4. Effect of partial NO2
NHþ
3 nutrition (100/0 NH4 /NO3 and 75/25 NH4 /NO3 ) on
4 accumulation in ‘Nanguang’
and ‘Elio’ rice cultivars at seedling stage, early tillering stage and maximal tillering stage in a hydroponic culture system (mg
pot21)
Cultivars
Leaves
‘Nanguang’
‘Elio’
Roots
‘Nanguang’
‘Elio’
2
NHþ
4 -N/NO3 -N
Seedling stage
Early tillering stage
Maximal tillering stage
100/0
75/25
100/0
75/25
4.33 + 0.10a
4.91 + 0.23b
7.97 + 0.07b
6.24 + 0.10a
13.3 + 0.21a
14.6 + 0.36a
22.5 + 1.20a
19.2 + 0.40a
36.1 + 0.45a
39.8 + 0.53b
58.5 + 1.57b
47.3 + 0.37a
100/0
75/25
100/0
75/25
0.65 + 0.01a
0.80 + 0.04b
1.08 + 0.06b
0.84 + 0.03a
2.00 + 0.01a
2.23 + 0.02b
2.79 + 0.01b
2.13 + 0.08a
4.98 + 0.29a
6.32 + 0.16b
6.75 + 0.45b
5.18 + 0.14a
2
Each value was the average of three replicates. Superscript letters show the statistical significance (P , 0.05) of the treatments (100/0 NHþ
4 /NO3 and
2
75/25 NHþ
4 /NO3 ) for a given organ in ‘Nanguang’ or ‘Elio’ cultivars.
Relative expression of OsAMT1s (OsAMT1;1 –1;3)
The expression level of OsAMT1;1 was unaltered in both
NHþ
4 -only and PNN nutrient solutions in leaves of
‘Nanguang’, but was depressed by 67 % in PNN nutrient
solution in ‘Elio’ (Fig. 3). However, PNN enhanced the
expression of OsAMT1;2 (184 % in ‘Nanguang’ and
57.9 % in ‘Elio’) while it depressed the expression of
OsAMT1;3 (75.1 % in ‘Nanguang’ and 62.5 % in ‘Elio’)
in leaves.
PNN increased the expressions of all three genes
(OsAMT1;1, OsAMT1;2 and OsAMT 1;3) in roots of both
cultivars, i.e. 19.8, 130 and 93.4 % in ‘Nanguang’, and
10.5, 164 and 49.2 % in ‘Elio’.
Expression amounts of OsAMT1;1, OsAMT1;2 and
OsAMT1;3 were increased by 15.1 % in roots of
‘Nanguang’, and 12.3 % in roots of ‘Elio’ by PNN. In
leaves of ‘Nanguang’ and ‘Elio’, PNN decreased
OsAMT1;1 expression by 0.50 and 10.3 %, improved
OsAMT1;2 expression by 1.87 and 0.56 %, and depressed
OsAMT1;3 expression by 1.99 and 2.28 %, respectively.
In summary, PNN improved expression of OsAMT1s by
14.5 % in ‘Nanguang’ and 0.29 % in ‘Elio’. The different
effect of PNN on the expression of OsAMT1s between the
two cultivars could be attributed to the different expression
pattern of OsAMT1;1 in leaves, which was unchanged in
‘Nanguang’ and decreased in ‘Elio’ by PNN treatment.
In total, the expression of OsAMT1s was 64.0 and 71.9 %
in the roots of ‘Nanguang’ and ‘Elio’, respectively, while in
the leaves the equivalent figures were 36.0 and 28.1 %. The
transcript levels of OsAMT1;1 were higher in both roots and
leaves (Fig. 3) than those of OsAMT1;2 and OsAMT1;3.
The expression of OsAMT1;2 and OsAMT1;3 was very
low in the roots. Therefore, the expression of OsAMT1s in
rice is mainly in roots, but a different expression pattern
of OsAMT1;1 in leaves was correlated with the response
of rice cultivars with differing NUE under PNN.
DISCUSSION
Rice is being increasingly cultivated under intermittent irrigation, or even in aerobic soil in which NO2
3 nutrition is
very important. On the other hand, low NUE by rice
leads not only to a heavy economic burden for farmers
but also to environmental pollution. In this study, the
relationship between PNN and NUE was investigated, in
order to clarify the mechanism of higher NUE under PNN.
Since Malavolta (1954) first reported a favourable effect
of NO2
3 on rice growth, several reports (Youngdahl et al.,
15
þ
2
þ
2
TA B L E 5. Effect of partial NO2
NHþ
3 nutrition (100/0 NH4 /NO3 and 75/25 NH4 /NO3 ) on
4 uptake efficiency in ‘Nanguang’
and ‘Elio’ rice cultivars at seedling stage, early tillering stage and maximal tillering stage in a hydroponic culture system (%)
Cultivars
Leaves
‘Nanguang’
‘Elio’
Roots
‘Nanguang’
‘Elio’
2
NHþ
4 -N/NO3 -N
Seedling stage
Early tillering stage
Maximal tillering stage
100/0
75/25
100/0
75/25
3.61 + 0.09a
5.45 + 0.26b
6.64 + 0.07a
6.93 + 0.25a
5.81 + 0.29a
8.10 + 0.20b
9.36 + 0.50a
10.7 + 0.45a
10.0 + 0.74a
14.7 + 0.36b
16.6 + 0.44a
17.5 + 0.14a
100/0
75/25
100/0
75/25
0.54 + 0.01a
0.88 + 0.02b
0.90 + 0.05a
0.93 + 0.03a
0.83 + 0.01a
1.16 + 0.01b
1.16 + 0.01a
1.18 + 0.05a
1.38 + 0.08a
2.34 + 0.06b
1.88 + 0.12a
1.92 + 0.05a
2
Each value was the average of three replicates. Superscript letters show the statistical significance (P , 0.05) of the treatments (100/0 NHþ
4 /NO3 and
2
/NO
)
for
a
given
organ
in
‘Nanguang’
or
‘Elio’
cultivars.
75/25 NHþ
4
3
1158
Duan et al. — Responses of Rice to Partial Nitrate Nutrition
TA B L E 6. Effects of NO2
3 on kinetic parameters; Vmax (maximum uptake rate) and Km (apparent Michaelis –Menten
constant), of 15NHþ
4 net uptake by ‘Nanguang’ and ‘Elio’ rice cultivars with different nitrogen use efficiencies at the seedling
stage
Vmax (mg g21 plant d.wt h21)
Km (mM)
Cultivars
Without NO2
3
With NO2
3
Without NO2
3
With NO2
3
‘Nanguang’
‘Elio’
51.1 + 0.21a
58.6 + 0.26a
58.3 + 0.27b
58.4 + 0.37a
30.2 + 2.07a
29.7 + 2.26a
31.1 + 1.54a
31.6 + 2.77a
2
Each value was the average of three replicates. Superscript letters show the statistical significance (P , 0.05) of the treatments (100/0 NHþ
4 /NO3 and
2
75/25 NHþ
/NO
)in
‘Nanguang’
or
‘Elio’
cultivars.
4
3
1982; Qian et al., 2004) have demonstrated that rice growth
and yield were superior in PNN as compared with in NHþ
4
alone. In the present study, PNN improved growth, N
accumulation, NHþ
4 uptake and OsAMT1 expression of
the high-NUE rice cultivar (‘Nanguang’). When compared
with that under solely NHþ
4 nutrition, dry matter and N
accumulation in ‘Nanguang’ were increased in PNN at
every growth stage. Grain yield of ‘Nanguang’ was
increased by PNN treatment, while that of the low-NUE
cultivar ‘Elio’ was similar in the two treatments.
However, N physiological use efficiency of ‘Nanguang’
did not change under PNN, and this suggested that the
improved NUE of ‘Nanguang’ by PNN might be attributed
to N uptake efficiency, but not N physiological use
efficiency.
Much research work has reported that growth and yield
maximization in a mixed N supply could be attributed to
an upregulation of N uptake and metabolism by NO2
3.
The present experiments using the 15N-label technique for
NHþ
4 uptake have shown that PNN significantly stimulated
NHþ
4 uptake by the ‘Nanguang’ cultivar from the seedling
to maximum tillering stage, and thus has improved the
2
NHþ
4 uptake efficiency. A stimulatory effect by NO3 on
þ
NH4 uptake was also recorded in rice and soybean
(Saravitz et al., 1994; Duan et al., 2006). Kronzucker
et al. (1999) reported that net NHþ
4 acquisition was
increased by as much as 50 % in PNN, compared with
the NHþ
4 -only supply. Results of kinetic studies have
shown that the improved NHþ
4 uptake rate by PNN was
mainly due to an increased Vmax, but no change in Km.
Since Vmax describes the number of ion transporters in
cell membranes while Km describes the affinity of the transporter for the ion, the improved NHþ
4 uptake by rice plants
2
under the partial replacement of NHþ
4 by NO3 could be
þ
attributed to the increased number of NH4 carriers.
All three OsAMT1 genes (OsAMT1;1, OsAMT1;2 and
OsAMT1;3) encode functional ammonium transporters
and play a key role in the influx of NHþ
4 from a low external
NHþ
4 concentration (Kumar et al., 2003). In the results presented here, OsAMT1;1 was expressed chiefly in roots and
leaves, while OsAMT1;2 and OsAMT1;3 were strongly
expressed in roots and only slightly expressed in leaves of
both cultivars, which was consistent with the results of
Sonoda et al. (2003).
F I G . 3. Relative OsAMT1;1 (1;1), OsAMT1;2 (1;2) and OsAMT1;3 (1;3) gene expression level (%) in roots and leaves of ‘Nanguang’ and ‘Elio’ rice
cultivars. For each gene, the relative amounts of mRNA in different organs and treatments were added together and then expressed as a percentage of
2
the sum, in ‘Nanguang’ and ‘Elio’ rice cultivars. Lower case letters show the statistical significance (P , 0.05) of the treatments (100/0 NHþ
4 /NO3
2
and 75/25 NHþ
/NO
)
for
gene
expression
in
‘Nanguang’
or
‘Elio’
cultivars.
4
3
Duan et al. — Responses of Rice to Partial Nitrate Nutrition
2
Under two different N cultivation systems with NHþ
4 /NO3
at either 100/0 or 75/25, the expression patterns of OsAMT1s
in the roots and shoots of two rice cultivars that differ in their
NUE were observed, and these results may be explained by
the results from the uptake studies. On the whole, PNN
improved the expression of OsAMT1 in the ‘Nanguang’ cultivar by 14.5 % but produced no change in expression in the
‘Elio’ cultivar, when compared with that value measured
under NHþ
4 -only supply. PNN improved the expression
of all three OsAMT1 genes in roots of both cultivars, by
15.1 % in ‘Nanguang’ and 12.3 % in ‘Elio’. In leaves, PNN
decreased the expression of OsAMT1;2 and OsAMT1;3 to a
very low level in both cultivars, but did not change the
expression of OsAMT1;1 in leaves of ‘Nanguang’. In contrast, PNN decreased OsAMT1;1 expression by 69 % in
leaves of ‘Elio’. Therefore, the differing responses of
OsAMT1;1 expression in leaves of the two cultivars under
PNN might have led to the changes in NHþ
4 uptake.
Therefore, it is suggested that the expression of OsAMT1
genes might be regulated, not only by NHþ
4 concentration,
but also by the form of N supplied. Furthermore, under
PNN, the NO2
3 concentration and the change in OsAMT1;1
expression in leaves of rice cultivars might be important
for their NUEs.
In the present study, OsAMT1s expression was investigated, and their relationship to NHþ
4 uptake was clarified.
However, there are still some low-affinity NHþ
4 transporters
and water channels; additionally there may be competition
from other ions, such as potassium (Kþ) (Schachtman and
Schroeder, 1994; Park et al., 1996; Santa-Maria et al.,
1997) that may affect NHþ
4 uptake by rice. However, the
molecular basis for the relationship between NHþ
4 uptake
þ
þ
and LAT, NHþ
4 and K , and NH4 and water channels is
at present unknown, and further work is needed to clarify
these issues.
In conclusion, PNN increased NHþ
transporter
4
(OsAMT1s) expression and NHþ
4 uptake, resulting in an
increased NHþ
4 uptake efficiency and biomass accumulation, and increased grain yield in the high-NUE rice cultivar ‘Nanguang’. In ‘Elio’, the low-NUE cultivar, the
changes were not observed under PNN. Therefore, the
increased NUE of ‘Nanguang’ could be attributed specifically to improved N uptake efficiency. The finding that
the rice cultivar with higher NUE had a more positive
response to PNN than that with a low NUE suggests that
there might be a close relationship between PNN and
NUE in rice.
AC KN OW L E DG E M E N T S
We thank Dr Tony Miller from Rothamsted Research, UK
both for his critical review of the contents and for his corrections to the English in this paper. This research work was
financially supported by the National Nature Science
Foundation of China (Nos 30671234 and 30390082),
National Basic Research and Development Program of
China (No. 2005CB120900) and Innovative Project for
Graduate Student in Jiangsu Province.
1159
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