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Ann Bot. 2007 Jun; 99(6): 1153–1160.
Published online 2007 Apr 11. doi:10.1093/aob/mcm051
PMCID: PMC3244343
PMID: 17428833
Y. H. Duan, Y. L. Zhang, L. T. Ye, X. R. Fan, G. H. Xu, and Q. R. Shen*
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Abstract
Background and Aims
There is increased evidence that partial nitrate (NO3−) nutrition (PNN) improves growth of rice (Oryza sativa), although the crop prefers ammonium (NH4+) to NO3− nutrition. It is not known whether the response to NO3− 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 NO3− supply in terms of dry weight, N accumulation, grain yield, NH4+ uptake and ammonium transporter expression [real-time polymerase chain reaction (PCR)].
Key Results
A ratio of 75/25 NH4+-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 NH4+-N/NO3−-N. Uptake experiments with 15N-NH4+ showed that NO3− increased NH4+ uptake efficiency in ‘Nanguang’ by increasing Vmax (14 %), but there was no effect on Km. This indicated that partial replacement of NH4+ by NO3− could increase the number of the ammonium transporters but did not affect the affinity of the transporters for NH4+. 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.
Key words: Ammonium transporter, partial NO3− nutrition, NH4+ uptake, nitrogen use efficiency, rice, Oryza sativa
INTRODUCTION
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 paddy field, ammonium (NH4+) 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 NO3− nutrition (PNN) of rice crops, and their results have shown that lowland rice was exceptionally efficient in absorbing NO3− 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 NO3− 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 NO3− uptake by lowland rice might be far more important than was previously thought; its uptake rate could be comparable with that of NH4+, 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 NH4+, rice roots are actually exposed to a mixed N supply in the rhizosphere (Briones et al., 2003; Y. L. Li et al., 2006).
When rice plants in solution culture were fed with a mixture of NH4+ 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 NO3− 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 increased influx of NH4+ by NO3− (Kronzucker et al. 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 low-affinity transport system (LAT) (Howitt and Udvardi, 2000; Loque and von Wiren, 2004). At low NH4+ concentration, uptake is mediated by HATs and exhibits sensitivity to metabolic inhibitors (Wang et al., 1993). At high NH4+ 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 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 NH4+ and N starvation (Suenaga et al., 2003; B. Z. Li et al., 2006). In contrast, the expression of OsAMT1s in response to NO3− 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 NH4+ uptake of two rice cultivars with differing NUEs were reinvestigated, and then the expression of OsAMT1s (OsAMT1;1–1;3) under NH4+ nutrition with and without NO3− was characterized. Finally a possible relationship between PNN and NUE was proposed
MATERIALS AND METHODS
Plant materials
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 Table1. ‘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.
Table1.
Characteristics of ‘Nanguang’ and ‘Elio’ rice cultivars evaluated in field experiments (N = 180 kg ha−1) in 2004 (Zhang et al., 2007)
| Cultivars | Nitrogen use efficiency (kg kg−1) | Grain yield (t ha−1) | Total biomass weight (t ha−1) | Growth duration (d) | Tillers/plant | Plant height (cm) | 1000-grain weight (g) |
|---|---|---|---|---|---|---|---|
| ‘Nanguang’ | 37 | 9·12 | 18·7 | 163 | 7·3 | 108 | 26·8 |
| ‘Elio’ | 30 | 7·83 | 13·8 | 157 | 3·4 | 96 | 39·7 |
All the hydroponic experiments in this study were carried out in the greenhouse with temperatures ranging from 20 °C at midnight to 35 °C 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 (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 subjected to two treatments of different NH4+-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 μm: Fe-EDTA, 20; MnCl2, 9·1; (NH4)6Mo7O24, 0·4; H3BO3, 37; ZnSO4, 0·8; CuSO4, 0·3. To inhibit nitrification, 7 µm dicyandiamide (DCD-C2H4N4) was mixed into all the solutions. The nutrient solution was renewed every 3 d. No NO3− 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 °C for half an hour to inactivate the enzymes, and finally dried to a constant weight at 70 °C. The dry weight was recorded. Nitrogen content in plants was determined by the Kjeldahl method (Chu et al., 2004).
15N-labelled growth experiment
Rice plants were cultivated as described above and treated with different N forms (100/0 and 75/25 of NH4+-N/NO3−-N). NH4+ was labelled by 15N [(NH4)2SO4, 10·7 % atom 15N 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 15N abundance in each fraction was determined using a MAT251 isotope mass spectrometer.
Kinetics of 15N-labelled NH4+ uptake
Rice seedlings with four leaves (30 d after germination) were prepared in a nutrient solution containing 1·43 mm 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 15NH4+ 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 NO3− on 15NH4+ uptake kinetics, 0·25 mm Ca(NO3)2 was added to the series of 15NH4+-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 15NH4+-containing solutions for 2 h at 30 ± 1 °C and a light intensity of 900 µmol photos m−2 s−1 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 NH4+ 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 to a partially replaced NH4+ solution (75/25 NH4+/NO3−) and the other half to an NH4+-only solution with the 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 °C until analysis.
Total RNA from 100 mg of plant material was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Approximately 2 µg 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 µl containing 1 × PCR buffer, 1 mm dNTPs, 0·5 µm oligo(dT) primer (Promega) and 0·5 U of RNase inhibitor (TaKaRa). The PCR amplification was performed using Takara Ex-Taq™ polymerase for target genes and actin.
For polymerase chain reaction (PCR), the primers (Table2) for OsAMT1;1–1;3 amplification were designed 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.
Table2.
Primers for OsAMT1;1, OsAMT1;2, OsAMT1;3 and Actin genes
| Target genes | GenBank accession no. | Direction | Sequence of primers |
|---|---|---|---|
| OsAMT1;1 | AF289477 | Forward | 5′-GGTCATCTTCGGGTGGGTCA-3′ |
| Reverse | 5′-CGTGCCGTGTCAGGTCCAT-3′ | ||
| OsAMT1;2 | AF289478 | Forward | 5′-GAAGCACATGCCGCAGACA-3′ |
| Reverse | 5′-GACGCCCGACTTGAACAGC-3′ | ||
| OsAMT1;3 | AF289479 | Forward | 5′-GCGAACGCGACGGACTA-3′ |
| Reverse | 5′-GACCTGTGGGACCTGCTTG-3′ | ||
| Actin | NM_197297 | Forward | 5′-TTATGGTTGGGATGGGACA-3′ |
| Reverse | 5′-AGCACGGCTTGAATAGCG-3′ |
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 µL containing: 0·5 µL of each primer (10 pmol L−1) for target genes or Actin (Table2), 10 µL of SYBR Green PCR master mix [TaKaRa Biotechnology (Dalina) Co., Ltd], 2 µL of cDNA and 7 µL of RNase-free water. The real-time PCR conditions were as follows: denaturation at 95 °C for 30 s; followed by 40 cycles at 95 °C for 10 s, 55 °C for 20 s, and 72 °C for 30 s; followed by 95 °C for 1 min and 55 °C 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).
where W(L + 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 = 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.).
RESULTS
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 NH4+ 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 (Figs1 and and2),2), suggesting that partial replacement of NH4+ by NO3− is more effective in early growth stages of rice plants.
Effect of partial NO3− nutrition (100/0 NH4+/NO3− and 75/25 NH4+/NO3−) on the dry weight (g pot−1) 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 (P < 0·05) of the treatments (100/0 NH4+/NO3− and 75/25 NH4+/NO3−) for a given growth stage in ‘Nanguang’ or ‘Elio’ cultivars.
Effect of partial NO3− nutrition (100/0 NH4+/NO3− and 75/25 NH4+ /NO3−) on the N accumulation (mg pot−1) 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 (P < 0·05) of the treatments (100/0 NH4+/NO3− and 75/25 NH4+/NO3−) for a given growth stage in ‘Nanguang’ or ‘Elio’ cultivars.
Grain yield and N physiological use efficiency
Grain yield of ‘Nanguang’ was higher than that of ‘Elio’ under NH4+-only cultivation (Table3). PNN led to a 21 % increase in grain yield in ‘Nanguang’ while there was no effect in ‘Elio’.
Table3.
Effect of partial NO3− nutrition (100/0 NH4+/NO3− 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 | NH4+/NO3− | Grain yield (g pot−1) | Physiological N use efficiency (%) |
|---|---|---|---|
| ‘Nanguang’ | 100/0 | 14·7 ± 1·07a | 18·5 ± 0·69a |
| 75/25 | 17·8 ± 0·82b | 18·8 ± 0·56a | |
| ‘Elio’ | 100/0 | 9·80 ± 0·76a | 12·6 ± 1·02a |
| 75/25 | 10·5 ± 0·88a | 13·3 ± 0·87a |
Each value was the average of three replicates. Superscript letters show the statistical significance (P < 0·05) of the treatments (100/0 NH4+/NO3− and 75/25 NH4+/NO3−) in ‘Nanguang’ or ‘Elio’ cultivars.
Nitrogen physiological use efficiencies of ‘Nanguang’ and ‘Elio’ were constant with or without NO3− (Table3), though the yield and N accumulation of ‘Nanguang’ were improved by PNN.
15NH4+ accumulation and uptake efficiency at early growth stages
PNN increased 15NH4+ 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 NH4+-only treatment (Table4). It was less effective in ‘Elio’ except at the early tillering stage.
Table4.
Effect of partial NO3− nutrition (100/0 NH4+/NO3− and 75/25 NH4+/NO3−) on 15NH4+ accumulation in ‘Nanguang’ and ‘Elio’ rice cultivars at seedling stage, early tillering stage and maximal tillering stage in a hydroponic culture system (mg pot−1)
| Cultivars | NH4+-N/NO3−-N | Seedling stage | Early tillering stage | Maximal tillering stage |
|---|---|---|---|---|
| Leaves | ||||
| ‘Nanguang’ | 100/0 | 4·33 ± 0·10a | 13·3 ± 0·21a | 36·1 ± 0·45a |
| 75/25 | 4·91 ± 0·23b | 14·6 ± 0·36a | 39·8 ± 0·53b | |
| ‘Elio’ | 100/0 | 7·97 ± 0·07b | 22·5 ± 1·20a | 58·5 ± 1·57b |
| 75/25 | 6·24 ± 0·10a | 19·2 ± 0·40a | 47·3 ± 0·37a | |
| Roots | ||||
| ‘Nanguang’ | 100/0 | 0·65 ± 0·01a | 2·00 ± 0·01a | 4·98 ± 0·29a |
| 75/25 | 0·80 ± 0·04b | 2·23 ± 0·02b | 6·32 ± 0·16b | |
| ‘Elio’ | 100/0 | 1·08 ± 0·06b | 2·79 ± 0·01b | 6·75 ± 0·45b |
| 75/25 | 0·84 ± 0·03a | 2·13 ± 0·08a | 5·18 ± 0·14a |
Each value was the average of three replicates. Superscript letters show the statistical significance (P < 0·05) of the treatments (100/0 NH4+/NO3− and 75/25 NH4+/NO3−) for a given organ in ‘Nanguang’ or ‘Elio’ cultivars.
15NH4+ uptake efficiency of ‘Nanguang’ was increased by PNN at all three growth stages, while no difference was observed in ‘Elio’ (Table5). The increase could be as high as 51 % in leaves at the seedling stage, and 70 % in roots at the maximal tillering stage.
Table5.
Effect of partial NO3− nutrition (100/0 NH4+/NO3− and 75/25 NH4+/NO3−) on 15NH4+ uptake efficiency in ‘Nanguang’ and ‘Elio’ rice cultivars at seedling stage, early tillering stage and maximal tillering stage in a hydroponic culture system (%)
| Cultivars | NH4+-N/NO3−-N | Seedling stage | Early tillering stage | Maximal tillering stage |
|---|---|---|---|---|
| Leaves | ||||
| ‘Nanguang’ | 100/0 | 3·61 ± 0·09a | 5·81 ± 0·29a | 10·0 ± 0·74a |
| 75/25 | 5·45 ± 0·26b | 8·10 ± 0·20b | 14·7 ± 0·36b | |
| ‘Elio’ | 100/0 | 6·64 ± 0·07a | 9·36 ± 0·50a | 16·6 ± 0·44a |
| 75/25 | 6·93 ± 0·25a | 10·7 ± 0·45a | 17·5 ± 0·14a | |
| Roots | ||||
| ‘Nanguang’ | 100/0 | 0·54 ± 0·01a | 0·83 ± 0·01a | 1·38 ± 0·08a |
| 75/25 | 0·88 ± 0·02b | 1·16 ± 0·01b | 2·34 ± 0·06b | |
| ‘Elio’ | 100/0 | 0·90 ± 0·05a | 1·16 ± 0·01a | 1·88 ± 0·12a |
| 75/25 | 0·93 ± 0·03a | 1·18 ± 0·05a | 1·92 ± 0·05a |
Each value was the average of three replicates. Superscript letters show the statistical significance (P < 0·05) of the treatments (100/0 NH4+/NO3− and 75/25 NH4+/NO3−) for a given organ in ‘Nanguang’ or ‘Elio’ cultivars.
Kinetic parameters of 15NH4+ net uptake
PNN increased the uptake rate (Vmax) of 15NH4+ by 14·1 % in ‘Nanguang’, while there was no change in ‘Elio’ (Table6), indicating that the number of transporters for NH4+ 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 NH4+ in rice roots.
Table6.
Effects of NO3− on kinetic parameters; Vmax (maximum uptake rate) and Km (apparent Michaelis–Menten constant), of 15NH4+ net uptake by ‘Nanguang’ and ‘Elio’ rice cultivars with different nitrogen use efficiencies at the seedling stage
| Vmax (μg g−1 plant d·wt h−1) | Km (μm) | |||
|---|---|---|---|---|
| Cultivars | Without NO3− | With NO3− | Without NO3− | With NO3− |
| ‘Nanguang’ | 51·1 ± 0·21a | 58·3 ± 0·27b | 30·2 ± 2·07a | 31·1 ± 1·54a |
| ‘Elio’ | 58·6 ± 0·26a | 58·4 ± 0·37a | 29·7 ± 2·26a | 31·6 ± 2·77a |
Each value was the average of three replicates. Superscript letters show the statistical significance (P < 0·05) of the treatments (100/0 NH4+/NO3− and 75/25 NH4+/NO3−)in ‘Nanguang’ or ‘Elio’ cultivars.
Relative expression of OsAMT1s (OsAMT1;1–1;3)
The expression level of OsAMT1;1 was unaltered in both NH4+-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.
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 the sum, in ‘Nanguang’ and ‘Elio’ rice cultivars. Lower case letters show the statistical significance (P < 0·05) of the treatments (100/0 NH4+/NO3− and 75/25 NH4+/NO3−) for gene expression in ‘Nanguang’ or ‘Elio’ cultivars.
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 NO3− 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 NO3− on rice growth, several reports (Youngdahl et al., 1982; Qian et al., 2004) have demonstrated that rice growth and yield were superior in PNN as compared with in NH4+ alone. In the present study, PNN improved growth, N accumulation, NH4+ uptake and OsAMT1 expression of the high-NUE rice cultivar (‘Nanguang’). When compared with that under solely NH4+ 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 NO3−. The present experiments using the 15N-label technique for NH4+ uptake have shown that PNN significantly stimulated NH4+ uptake by the ‘Nanguang’ cultivar from the seedling to maximum tillering stage, and thus has improved the NH4+ uptake efficiency. A stimulatory effect by NO3− on NH4+ uptake was also recorded in rice and soybean (Saravitz et al., 1994; Duan et al., 2006). Kronzucker et al. (1999) reported that net NH4+ acquisition was increased by as much as 50 % in PNN, compared with the NH4+-only supply. Results of kinetic studies have shown that the improved NH4+ 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 NH4+ uptake by rice plants under the partial replacement of NH4+ by NO3− could be 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 NH4+ from a low external NH4+ 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).
Under two different N cultivation systems with NH4+/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 NH4+-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 NH4+ uptake. Therefore, it is suggested that the expression of OsAMT1 genes might be regulated, not only by NH4+ concentration, but also by the form of N supplied. Furthermore, under PNN, the NO3− 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 NH4+ uptake was clarified. However, there are still some low-affinity NH4+ transporters and water channels; additionally there may be competition from other ions, such as potassium (K+) (Schachtman and Schroeder, 1994; Park et al., 1996; Santa-Maria et al., 1997) that may affect NH4+ uptake by rice. However, the molecular basis for the relationship between NH4+ uptake and LAT, NH4+ and K+, and NH4+ and water channels is at present unknown, and further work is needed to clarify these issues.
In conclusion, PNN increased NH4+ transporter (OsAMT1s) expression and NH4+ uptake, resulting in an increased NH4+ 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.
ACKNOWLEDGEMENTS
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.
LITERATURE CITED
- Briones AM, Jr, Okabe S, Umemiya Y, Ramsing NB, Reichardt W, Okuyama H. Ammonia-oxidizing bacteria on root biofilms and their possible contribution to N use efficiency of different rice cultivars. Plant and Soil. 2003;250:335–348. [Google Scholar]
- Chu GX, Shen QR, Cao JL. Nitrogen fixation and N transfer from peanut to rice cultivated in aerobic soil in an intercropping system and its effect on soil N fertility. Plant and Soil. 2004;263:17–27. [Google Scholar]
- Cox WJ, Reisenauer HM. Growth and ion uptake by wheat supplied nitrogen as nitrate, or ammonium, or both. Plant and Soil. 1973;38:363–380. [Google Scholar]
- De Macale MAR, Velk PLG. The role of Azolla cover in improving the nitrogen use efficiency of lowland rice. Plant and Soil. 2004;263:311–321. [Google Scholar]
- Duan YH, Zhang YL, Shen QR, Wang SW. Nitrate effect on rice growth and nitrogen absorption and assimilation at different growth stages. Pedosphere. 2006;16:707–717. [Google Scholar]
- Duan YH, Yin XM, Zhang YL, Shen QR. Mechanisms of nitrate enhancement on growth and nitrogen uptake by rice. Pedosphere. 2007 (in press) [Google Scholar]
- Eisenthal R, Cornish-Bowden A. The direct linear plot, a new graphical procedure for estimating enzyme kinetic parameters. Biochemical Journal. 1974;139:715–720. [PMC free article] [PubMed] [Google Scholar]
- Heberer JA, Below FE. Mixed nitrogen nutrition and productivity of wheat grown in hydroponics. Annals of Botany. 1989;63:643–649. [Google Scholar]
- Howitt SM, Udvardi MK. Structure, function and regulation of ammonium transporters in plants. Biochimica et Biophysica Acta. 2000;1465:152–170. [PubMed] [Google Scholar]
- Jain M, Tyagi AK, Khurana JP. Genome-wide analysis, evolutionary expansion, and expression of early auxin-responsive SAUR gene family in rice (Oryza sativa) Genomics. 2006;88:360–371. [PubMed] [Google Scholar]
- Kirk GJD. Plant-mediated processes to acquire nutrients: nitrogen uptake by rice plants. Plant and Soil. 2001;232:129–134. [Google Scholar]
- Kirk GJD, Kronzucker HJ. The potential for nitrification and nitrate uptake in the rhizosphere of wetland plants: a modelling study. Annals of Botany. 2005;96:639–646. [PMC free article] [PubMed] [Google Scholar]
- Kronzucker HJ, Siddiqi MY, Glass ADM, Kirk GJD. Nitrate–ammonium synergism in rice: a subcellular flux analysis. Plant Physiology. 1999;119:1041–1045. [PMC free article] [PubMed] [Google Scholar]
- Kronzucker HJ, Glass ADM, Siddiqi MY, Kirk GJD. Comparative kinetic analysis of ammonium and nitrate acquisition by tropical lowland rice: implications for rice cultivation and yield potential. New Phytologist. 2000;145:471–476. [Google Scholar]
- Kumar A, Silim SN, Okamoto M, Siddiqi MY, Glass ADM. Differential expression of three members of the AMT1 gene family encoding putative high-affinity NH4+ transporters in roots of Oryza sativa subspecies indica. Plant, Cell and Environment. 2003;26:907–914. [PubMed] [Google Scholar]
- Ladha JK, Kirk GJD, Bennett J, Peng S, Reddy CK, Reddy PM, Singh U. Opportunities for increased nitrogen use efficiency from improved lowland rice germplasm. Field Crops Research. 1998;56:41–71. [Google Scholar]
- Lauter FR, Ninnemann O, Bucher M, Riesmeier JW, Frommer WB. Preferential expression of an ammonium transporter and of two putative nitrate transporters in root hairs of tomato. Proceedings of the National Academy of Sciences of the USA. 1996;93:8139–8144. [PMC free article] [PubMed] [Google Scholar]
- Li BZ, Xin WJ, Sun SB, Shen QR, Xu GH. Physiological and molecular responses of nitrogen-starved rice plants to re-supply of different nitrogen sources. Plant and Soil. 2006;287:145–159. [Google Scholar]
- Li YL, Zhang YL, Hu J, Shen QR. Contribution of nitrification happened in rhizospheric soil growing with different rice cultivars to N nutrition. Biology and Fertility of Soils. 2006;43:417–425. [Google Scholar]
- Loque D, von Wiren N. Regulatory levels for the transport of ammonium in plant roots. Journal of Experimental Botany. 2004;55:1293–1305. [PubMed] [Google Scholar]
- Malavolta E. Studies on the nitrogenous nutrition of rice. Plant Physiology. 1954;29:98–99. [PMC free article] [PubMed] [Google Scholar]
- Matt P, Geiger M, Walch-Liu P, Engels C, Krapp A, Stitt M. Elevated carbon dioxide increases nitrate uptake and nitrate reductase activity when tobacco is growing on nitrate, but increases ammonium uptake and inhibits nitrate reductase activity when tobacco is growing on ammonium nitrate. Plant, Cell and Environment. 2001;24:1119–1137. [Google Scholar]
- Moll RH, Kamprath EJ, Jackson WA. Analysis and interpretation of factors which contribute to efficiency of nitrogen utilization. Agronomy Journal. 1982;74:562–564. [Google Scholar]
- Ninnemann O, Jauniaux JC, Frommer WB. Identification of a high affinity ammonium transporter from plants. EMBO Journal. 1994;13:3464–3471. [PMC free article] [PubMed] [Google Scholar]
- Park JH, Saier M. Characterization of the MIP family of transmembrane channel proteins. Journal of Membrane Biology. 1996;153:171–180. [PubMed] [Google Scholar]
- Pearson JN, Finnemann J, Schjoerring JK. Regulation of the high-affinity ammonium transporter (BnAMT1;2) in the leaves of Brassica napus by nitrogen status. Plant Molecular Biology. 2002;49:483–490. [PubMed] [Google Scholar]
- Qian XQ, Shen QR, Xu GH, Wang JJ, Zhou MY. Nitrogen form effects on yield and nitrogen uptake of rice crop grown in aerobic soil. Journal of Plant Nutrition. 2004;27:1061–1076. [Google Scholar]
- Raman DR, Spanswick RM, Walker LP. The kinetics of nitrate uptake from flowing nutrient solutions by rice: influence of pretreatment and light. Bioresource Technology. 1995;53:125–132. [Google Scholar]
- Salvemini F, Marini AM, Riccio A, Patriarca EJ, Chiurazzi M. Functional characterization of an ammonium transporter gene from. Lotus japonicus. Gene. 2001;270:237–243. [PubMed] [Google Scholar]
- Santa-Maria GE, Rubio F, Dubcovsky J, Rodriguez-Navarro A. The HAK1 gene of barley is a member of a large gene family and encodes a high-affinity potassium transporter. Plant Cell. 1997;9:2281–2289. [PMC free article] [PubMed] [Google Scholar]
- Saravitz CH, Chaillous S, Musset J. Influence of nitrate on uptake of ammonium by nitrogen-depleted soybean: is the effect located in roots or shoots? Annals of Botany. 1994;45:1575–1584. [Google Scholar]
- Schachtman DP, Schroeder JL. Strcutre and transport mechanism of a high-affinity potassium uptake transporter from higher plants. Nature. 1994;370:655–658. [PubMed] [Google Scholar]
- Sheehy JE, Mnzava M, Cassman KG, Mitchell PL, PablicoLP, Robles RP, Ferrer A. Uptake of nitrogen by rice studied with a 15N point-placement technique. Plant and Soil. 2004;259:259–265. [Google Scholar]
- Simon-Rosin U, Wood CC, Udvardi MK. Molecular and cellular characterization of LjAMT2;1, an ammonium transporter from the model legume Lotus japonicus. Plant Molecular Biology. 2003;51:99–108. [PubMed] [Google Scholar]
- Sonoda Y, Ikeda A, Saiki S, von Wiren N, Yamaya T, Yamaguchi J. Distinct expression and function of three ammonium transporter genes (OsAMT1;1–1;3) in rice. Plant and Cell Physiology. 2003;44:726–734. [PubMed] [Google Scholar]
- Suenaga A, Moriya K, Sonoda Y, Ikeda A, von Wiren N, Hayakawa T, et al. Constitutive expression of a novel-type ammonium transporter OsAMT2 in rice plants. Plant and Cell Physiology. 2003;44:206–211. [PubMed] [Google Scholar]
- Tsuchiya T, Takesawa T, Kanzaki H, Nakamura I. Genomic structure and differential expression of two tandem-arranged GSTZ genes in rice. Gene. 2004;335:141–149. [PubMed] [Google Scholar]
- Wang MY, Siddeqi MY, Ruth TJ, Glass ADM. Ammonium uptake by rice roots. I. Kinetics of 13NH4+ influx across the plasmalemma. Plant Physiology. 1993;103:1259–1267. [PMC free article] [PubMed] [Google Scholar]
- Wang MY, Glass ADM, Shaff JE, Kochian LV. Ammonium uptake by rice roots. II. Electrophysiology. Plant Physiology. 1994;104:899–906. [PMC free article] [PubMed] [Google Scholar]
- von Wiren N, Lauter FR, Ninnemann O, Gillissen B, Walch-Liu P, Engels C, et al. Differential regulation of three functional ammonium transporter genes by nitrogen in root hairs and by light in leaves of tomato. Plant Journal. 2000;21:167–175. [PubMed] [Google Scholar]
- Yoshida S, Forno DA, co*ck JH, Gomez KA. Laboratory manual for physiological studies of rice. 2nd edn. The Philippines: International Rice Research Institute (IRRI); 1972. [Google Scholar]
- Youngdahl LJ, Pacheco R, Street JJ, Vlek PLG. The kinetics of ammonium and nitrate uptake by young rice plants. Plant and Soil. 1982;69:225–232. [Google Scholar]
- Yuko N, Fumiko F, Kanako S, Akiko H, Zenpei S, Junshi Y, Naoki K, Shoshi K. Validation of rice cDNA-based microarray data by the real-time RT–PCR methods. Miscellaneous Publication of the National Institute of Agrobiological Sciences. 2004;3:17–33. [Google Scholar]
- Zhang YH, Zhang YL, Shen QR. Nitrogen accumulation and translocation of different Japonica rice cultivars under different nitrogen application rates. Pedosphere. 2007 (in press) [Google Scholar]
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