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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

# The Author 2007. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: [email protected]

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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

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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

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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

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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

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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


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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

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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.

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