Journal of Experimental Botany, Vol. 58, No. 7, pp.

1729–1740, 2007
doi:10.1093/jxb/erm033 Advance Access publication 9 March, 2007
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

RESEARCH PAPER

Comparing nitrate storage and remobilization in two rice

cultivars that differ in their nitrogen use efficiency

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Xiaorong Fan1, Lijun Jia1, Yilin Li1, Susan J. Smith2, Anthony J. Miller2,* and Qirong Shen1,*
1
College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095, PR China
2
Crop Performance and Improvement Division, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK

Received 4 October 2006; Revised 2 January 2007; Accepted 25 January 2007

Abstract under both nitrate supply and deprivation conditions.

Checking NR gene expression showed that leaf ex-
Soil nitrogen (N) is available to rice crops as either
pression of OsNia1 was faster to respond to nitrate
nitrate or ammonium, but only nitrate can be accrued
deprivation than OsNia2 in both cultivars. These
in cells and so factors that influence its storage and
measurements are discussed in relation to cultivar
remobilization are important for N use efficiency
differences and physiological markers for NUE in rice.
(NUE). The hypothesis that the ability of rice crops to
remobilize N storage pools is an indicator of NUE was Key words: Cellular nitrate activities, nitrate reductase,
tested. When two commonly grown Chinese rice nitrogen use efficiency, rice, vacuolar remobilization.
cultivars, Nong Ken (NK) and Yang Dao (YD), were
compared in soil and hydroponics, YD had signifi-
cantly greater NUE for biomass production. The ability
of each cultivar to remobilize nitrate storage pools 24 h Introduction
after N supply withdrawal was compared. Although Ammonium is the main nitrogen (N) form available to
microelectrode measurements of the epidermal sub- rice roots growing under anaerobic paddy conditions, but
cellular nitrate pools in leaves and roots showed in aerobic and upland soils nitrate is the main N source.
similar patterns of vacuolar remobilization in both Rice (Oryza sativa L.) grows well in both mixed and
cultivars, whole-tissue analysis showed very little single source nitrate and ammonium supplies (Chanh
depletion of storage pools after 24 h. However, leaf et al., 1981; Youngdahl et al., 1982). In common with
epidermal cell cytosolic nitrate activities were signifi- most plants, rice can accumulate nitrate but not ammo-
cantly higher in YD when compared with NK. Before N nium within its tissues and this N store may be important
starvation and growing in 10 mM nitrate, the xylem for later growth and grain filling. For cultivation, rice is
nitrate activity in YD was lower than that of NK. After usually first grown in aerobic nursery soil beds where
24 h of N starvation the xylem nitrate had decreased nitrate is the main form of available N. Later japonica
more in YD than in NK. Tissue analysis of stems rice seedlings are transplanted into flooded soil that is
showed that YD had accumulated significantly more anaerobic and the plant is then chiefly supplied with
nitrate than NK, and the remobilization pattern sug- ammonium as an N source. Rice of the indica type can be
gested that this store is important for both cultivars. continuously grown in aerobic soils and therefore has
Changes in nitrate reductase activity (NRA) and ex- access to nitrate that may be stored in the leaves. Both
pression were measured. Growing in 10 mM nitrate, these types of rice can accumulate nitrate in the leaf when
NRA was undetectable in roots of both cultivars, and supplied with this N form (Fan et al., 2005) but it was felt
the leaf total NRA of equivalent leaves was similar in necessary to test if japonica rice has a greater capacity to
NK and YD. When the N supply was withdrawn, after store, and subsequently remobilize, vacuolar stored nitrate.
24 h NRA in NK was reduced to 80% but no decrease The term N use efficiency (NUE) can be defined
was found in YD. The proportion of NRA in an active in several different ways and it is likely to be regulated
form in YD was significantly higher than that in NK by many different genes (reviewed by Gallais and Hirel,

* To whom correspondence should be addressed. E-mail: tony.miller@bbsrc.ac.uk or shenqirong@njau.edu.cn

ª 2007 The Author(s).

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which
permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
1730 Fan et al.
2004; Good et al., 2004). During grain filling the ability expression (OsNRT1.1 and OsNRT2.1) was different in
of a plant to remobilize leaf-stored N is an important the two cultivars (Fan et al., 2005). These cultivars were
factor for NUE in crops, and has been strongly implicated chosen for this further study, first to compare their NUE
in quantitative trait locus (QTL) studies with cereals and then to measure how their physiological properties
(Mickelson et al., 2003). However, much less is known differ.
about NUE during the earlier vegetative stages of cereal
development when biomass is accumulating. Yet this growth
stage is important because it occurs when rainfall is heavy
and leaching losses are maximal. In cells, vacuolar nitrate Materials and methods

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may provide a store that maintains cellular assimilation Plant material
and thereby optimizes N utilization. Remobilization of Rice seeds of the cultivars Nong Ken (NK, japonica) and Yang Dao
vacuolar nitrate stores can be measured by removing all N (YD, indica) were surface sterilized in 3% (v/v) H2O2 for 10 min,
supply from a growing plant (van der Leij et al., 1998) then rinsed and and allowed to imbibe for 48 h in aerated distilled
and in this work this treatment has been used to test the water maintained at 30
C. After a further 24 h, germinated seeds
response of two rice cultivars that differ in their NUE. were placed in 400 ml pots and cultivated in a 30
C controlled-
environment chamber, with a 15 h day and a relative humidity at
Leaf tissue or sap nitrate concentrations are used as 6565%. Rice seedlings were grown in quarter-strength IRRI rice
indicators of a plant’s N status and this fact is exploited nutrient solution (pH 5.5) for 4 weeks arranged in a randomized
by farmers when making decisions on fertilizer applica- block design. Nitrogen was supplied as Ca(NO3)2 at 0.5 mM for the
tion rates (Schepers et al., 1992). Measurements of leaf first 2 weeks and then, unless stated otherwise, 5 mM was used for
tissue nitrate primarily determine nitrate stored in the the final 2 weeks (in some experiments 0.5 mM or 1.25 mM was
used). Other nutrients were added as follows: 2 mM K2SO4, 2 mM
vacuole. However, vacuolar nitrate accumulation within MgSO4, 1 mM CaCl2, 0.3 mM NaH2PO4, 40 lM Fe-EDTA, 9 lM
cereal leaf cells differs; the highest concentrations accu- MnCl2, 25 lM (NH4)6Mo7O24, 20 lM H3BO3, 1.5 lM ZnSO4, and
mulate in epidermal cells (Fricke et al., 1994). Barley root 1.5 lM CuSO4. Diluted HCl and NaOH were added to maintain
epidermal and cortical cell vacuolar nitrate can be a pH of 5.5 and this was monitored daily using a hand-held pocket-
remobilized during times of N deficiency and this source size pH meter (model 868, Thermo Orion, USA). Nutrient solutions
were replaced with fresh solution every 2 d.
can maintain cytosolic nitrate concentrations in the short
term (van der Leij et al., 1998). Some recent papers have
suggested that there is a close link between cytosolic Whole tissue N, nitrate, and NRA
nitrate activity and nitrate reductase activity (NRA). For The whole roots were used for tissue nitrate analysis. Mid-leaf
example, in leaf cells of Arabidopsis (Cookson et al., sections, 4 cm long, were cut from the first two fully expanded
2005) and barley root cells (Fan et al., 2006), changes in leaves and used for nitrate, NRA, and gene expression analysis. For
cytosolic nitrate activity could be measured under con- stem analysis, all the green tissue from the top of the root to the
fourth leaf ligule was used. All this plant tissue was sampled 5 h
ditions when cellular NRA was altered. As cytosolic into the light period. For nitrate analysis, the leaf or root tissue was
nitrate activity is important for determining the thermody- frozen in liquid N and then 1 g of the tissue was finely ground using
namic gradients for transport to and from the vacuole a pestle and mortar; the resulting powder was then extracted with
(Miller and Smith, 1992; De Angeli et al., 2006) how 20 ml of deionized, distilled water. This mixture was boiled for 30 min
NRA and mRNA expression changed during the remobi- and made up to 100 ml when it was cool. The mixture was filtered
through a filter paper (Whatman no. 2, 9 cm) and, for the shoot
lization of stored nitrate has also been examined. material, before it was filtered 0.2 g of active carbon was added
In order to study the relationship between NUE and to eliminate the effect of the chlorophyll. Nitrate analysis of the
key steps determining N distribution within the plant such supernatant was performed using a continuous-flow autoanalyser
as vacuolar storage and cytosolic nitrate activity these (model Autoanalyser 3, Bran & Luebbe, Germany). For total N
parameters were compared in two crop cultivars. Two rice analysis, 0.1 g of oven-dried and ground tissue samples was
weighed into 100 ml Kjeldahl digestion flasks. To each flask was
cultivars were used for these measurements because this added 5 ml of concentrated H2SO4 and the flask was gently heated.
species shows large variation in NUE (Koutroubas and When frothing had ceased, the heat was increased to 280
C and
Ntanos, 2003; Peng et al., 2006). Rice cultivation is then 30% H2O2 (v/v) was added to the flask intermittently until the
particularly wasteful as large amounts of applied N digest cleared. After complete digestion the flask was allowed to
fertilizer are lost into the surrounding environment (Vlek cool and water was slowly added to make up the volume to 100 ml.
A 5 ml sample was then analysed for total N using a continuous-
and Byrnes, 1986). The rice cultivars used in this study flow autoanalyser (model Autoanalyser 3, Bran & Luebbe).
have differing levels of N accumulation efficiency when NRA was measured using a modified method based on that
grown in soil or hydroponics with either nitrate or reported previously (Botrel and Kaiser, 1997). Frozen tissue
ammonium. In an earlier study, two Chinese rice cultivars samples (about 1.0 g) were ground to a fine powder using a chilled
were shown to have differing nitrate uptake rates when mortar and pestle in the presence of acid-washed sand. Samples
were homogenized with 5 ml of extraction buffer. Extraction buffers
supplied with 1 mM nitrate: Nong Ken (NK, japonica) contained 50 mM HEPES-KOH (pH 7.6), 10 mM cysteine, and
taking up less than Yang Dao (YD, indica) (Fan et al., 10 mM MgCl2 (for NRAact) or 5 mM EDTA (for NRAmax).
2005). Furthermore, the pattern of nitrate transporter The homogenates were centrifuged at 30 000 g for 15 min, and
 Nitrate storage and remobilization in rice 1731
the supernatants were assayed for NADH-NRA (Botrel and Kaiser, Xylem sap samples
1997). Xylem nitrate concentration can vary with the time of day and the
part of the plant which is sampled (Siebrecht et al., 2003) so care
Comparison of N use between two rice cultivars was taken to avoid these possible sources of error. The stems of
These experiments were conducted in soil in a controlled environ- 4-week-old rice seedlings were cut 5 cm below the uppermost leaf
ment cabinet at 28
C with a 16 h photoperiod with a photon flux node (ligule). Sap samples exuding from this cut surface were
density of 460–490 lmol m
2 s
1. Soil was analysed and the basic collected from 1900 h to 0700 h (i.e. 12 h overnight) by absorption
composition was as follows: organic matter 28.9 mg g
1, total N into a cotton wool pad (0.4 g) placed on the cut surface. The cotton
1.2 mg g
1, NH+4 -N 1.2 mg kg
1, NO

1
3 -N 7.4 mg kg , pH
wool was inside the base of a 25 ml centrifuge tube that was then
(water:soil, 1:2.5) 6.1, and clay size (<2 lm). The soil was air dried inverted over the top of the plant. Parafilm was placed around the

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and sieved (1 mm pore size), and urea (30 mg.N kg
1) and K2HPO4 plant–tube interface to minimize evaporation of the sap sample.
(93 mg kg
1) were mixed into it. Each pot was filled with 600 g of After 12 h exudation, the cotton wool containing the sap sample
soil, and three seedlings were planted in each. The pot was flooded was placed in a 5 ml Gilson disposable tip and then extracted using
with water to 1 cm depth 3 weeks after germination, and this was a Beckmann centrifuge model JA-21 (12 096 g for 5 min) with the
maintained at this level throughout the growth of the rice plants. tip down in a 50 ml centrifuge tube. After this time of centri-
Three replicate pots were used and all plant samples were harvested fugation, all the sap was extracted from the cotton wool (this was
4 h after the start of the light period. Tissue N analysis of the rice established by preliminary experiments; data not shown). Sap sample
plants was then used to determine the amounts accumulated by the volumes ranged from approximately 1 ml to 0.4 ml. Nitrate-
plant and then this was expressed relative to the whole plant dry selective microelectrodes were used to measure the nitrate activity
biomass (see Koutroubas and Ntanos, 2003). in these sap samples.
Plants were harvested at 44, 51, and 58 d after germination; these
plants were older than those grown in hydroponic culture.
Semi-quantitative RT-PCR of OsNia1 and OsNia2
Nitrate-selective microelectrodes Semi-quantitative RT-PCR was used to evaluate the level of nitrate
Double-barrelled nitrate-selective microelectrodes were prepared, reductase gene expression in roots and leaves. Total RNA was
calibrated, and used to record from root cells using the method isolated from rice tissue using TRIZOL reagent (Invitrogen). Two
described previously (Miller and Zhen, 1991; Zhen et al., 1991). milligrams of total RNA from each source was reverse-transcribed
For these microelectrode impalements a single primary root (not using M-MLV reverse transcriptase (Promega, Madison, WI, USA) and
seminal) of intact rice plants was held in a Plexiglas chamber oligo(dT) 30 primers (AAGCAGTGGTAACAACGCAGAGTAC
(volume 2.0 ml) and washed with a flowing solution (containing (T)30N-1 (Promega, Madison, WI, USA). OsActin (ActF, 5#-
5 mM MES, 0.5 mM CaCl2, and 0.05 mM KCl at pH 6.0) at TTATGGTTGGGATGGGACA-3#; ActR, 5#-AGCACGGCTT-
a flow rate of 1 ml min
1 as described previously (Walker et al., GAATAGCG-3#) was a standard reference to normalize the
1995). Nitrate was added as Ca(NO3)2 to the solution. The chamber quantity of total RNA used in each sample in the following cycle
was mounted on the stage of an Olympus microscope (model SZX9). conditions: 95
C for 2 min followed by 94
C for 30 s, 55
C for
Microelectrodes were prepared using filamented double-barrelled 30 s, and 72
C for 30 s, using a Bio-RAD cycler. The cycle
borosilicate glass as described previously (Zhen et al., 1991) number that corresponded to the linear phase of this PCR was
mounted on a micromanipulator (model NMN-21, Narashige, identified as described previously (Orsel et al., 2002) and 25 cycles
Japan). All root microelectrode measurements were obtained from were used in subsequent PCRs for all treatments to identify
mature cells 1–2 cm from the root tip (Zhen et al., 1991). Both variation in the cDNA product yield. The appropriate volume
microelectrode reference barrels and reference electrodes were back- needed to standardize each treatment relative to that of the test
filled with 100 mM KCl. Only electrode recordings which gave treatment was determined empirically and used in all subsequent
identical calibration curves before and after the cell measurements PCR. Two cytosolic nitrate reductase genes OsNia1 and OsNia2
were considered acceptable. For leaves, a different type of Plexiglas were PCR amplified using the standardized volumes. The specific
chamber was used with a sloping wall. In the middle of the wall primer sets for OsNia1 (AK102363) encoding the NADH-specific
there was a 2.53130.5 cm hole and a tie to hold the tissue in place NR (EC1.7.1.1 rice) and OsNia2 (AK102178) putatively encoding
during the recording. This design was modified from a chamber the NAD(P)H-bispecific NR (BlastX EC1.7.1.2 (formerly
described previously (Miller et al., 2001; Cookson et al., 2005). EC1.6.6.2) HvNia7 barley) were Nia1-F, 5#-CCAATTCTTT-
The electrophysiology solution covered half of the leaf area, and the CATCGTGTTCT-3#; Nia1-R, 5#-CATGCAGCATTTCGTTTCT-3#;
site for impaling was just above the liquid surface. The chamber and Nia2-F, 5#-GGAGGACGGGTGGGAGTA-3#; Nia2-R, 5#-
was placed on the stage of a dissecting microscope (model SZX9, TTCAGAAGACGAGGCAGGAC-3#, respectively. The PCR condi-
Olympus, Japan) for the recordings and the plant was intact for the tions were used for OsNia1, OsNia2, and OsActin. The approximate
measurement but the leaf was placed in the chamber for at least 1 h product sizes were 190 bp for OsNia1, 150 bp for OsNia2, and
before impalement. The photon flux density of photosynthetically 290 bp for OsActin. Amplified fragments were analysed by electro-
active radiation at the leaf surface was around 300 lmol m
2 s
1, phoresis on 1.5% agarose gels and visualized by staining with
measured using the Photosynthetic Determination System, ZID310 ethidium bromide. To check further that the amplified products were
(Harvard Centre for International Development, USA). The leaf was correct, fragments were then cloned in PMD18-T vector (TaKaRa)
illuminated by the microscope light throughout the microelectrode and sequenced. The nucleotide sequences were compared with those
recording and under these conditions, although a change in the released sequences in GenBank using the BLAST program.
membrane potential could be measured during light/dark transitions,
no change in cytosolic nitrate could be detected (data not shown),
confirming previously reported results for Arabidopsis leaf epider- Statistical analysis of data
mal cells (Cookson et al., 2005). Only fully expanded cells of the Two-way ANOVA and the t-test were used to compare data for
upper leaf epidermis in the middle of the leaf were used for the the two cultivars and for before and after N starvation using the
nitrate microelectrode measurements. This was the same region of Genstat software (GenStat 7th edition, Lawes Agricultural Trust,
the leaf that was used for tissue nitrate and NRA assays (see above). Rothamsted Research, UK).
1732 Fan et al.
Results
First it was confirmed that there are significant differences
in N utilization efficiency by the two cultivars. For these
measurements the plants were grown in soil that was later
flooded according to the traditional method of rice culti-
vation (see Materials and methods). These measurements
showed that throughout the period 44–58 d the cultivar YD
consistently showed significantly greater NUE for biomass

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accumulation when compared with NK (Fig. 1A). Simi-
larly, in hydroponic culture, YD also showed greater NUE
than NK at both 1 mM and 10 mM nitrate supply (Fig.
1B). In addition, biomass NUE for both cultivars signi-
ficantly increased at 10 mM nitrate when compared with
nitrate supplied at 1 mM. However, the increase in NUE
at 10 mM over that at 1 mM was not significantly different
between cultivars (Fig. 1B). Additional experiments com-
paring the two cultivars in hydroponic culture supplied
with a mixed nitrate and ammonium supply (1.25 mM
NH4NO3) also showed YD to be always better at N inter-
ception and use (data not shown).
The two rice cultivars were grown in hydroponic culture
for 4 weeks in either a mixed N supply (5 mM NH4NO3)
or in 10 mM nitrate [5 mM Ca(NO3)2] to compare bio-
mass production. The root and shoot dry weights are shown
in Fig. 2. The data showed that the seedlings produced
similar amounts of root and whole plant biomass that were
not significantly different when supplied with the same
amounts of N, and this growth did not depend on the form
of the nitrate supply, nitrate or ammonium (Fig. 2).
However, in 10 mM nitrate NK shoots produced signifi-
cantly less biomass than YD shoots growing in N supplied Fig. 1. A comparison of nitrogen use efficiency between two rice
cultivars growing in soil and in hydroponic culture. Nitrogen use
either as nitrate or mixed (P <0.05). efficiency was calculated as the percentage of N content of the plants
After growing the rice plants in 10 mM nitrate for 14 d expressed over the total N supplied to the plant (see Materials and
all external N supply was withdrawn. Figure 3 shows the methods for details). (A) Plants growing in soil; (B) plants (28 d old)
growing in hydroponic culture with either 1 mM or 10 mM nitrate
time course for changes in tissue nitrate during this N supply.
starvation and, during this 24 h period, the pattern of
tissue nitrate remobilization was different in each cultivar.
Tissue nitrate concentrations ranged from only 10 mM to and leaf cells of rice seedlings. Figure 4 shows an
30 mM (Fig. 3). In roots of both cultivars there was little example of a microelectrode recording obtained from
change in tissue nitrate, only the last measurement after a root rhizodermal cell. In this measurement the tip first
24 h starvation showed a small significant decrease in YD records a nitrate value of around 65 mM and the
that was not measured in NK. In the leaves a more membrane potential was around
90 mV. After 15 min
complicated pattern was found that showed no significant the nitrate value had slowly changed to a lower value of
change at the end of this 24 h period in either cultivar. around 3 mM and the membrane potential had become
The pattern of these changes was complicated by the more negative at
110 mV. This recording was unusual
diurnal changes in tissue nitrate pools (Steingrover et al., because the measurement was obtained from two different
1986) and, for this reason, the light/dark periods are compartments of the same cell and these values corre-
shown (Fig. 3). However, in the leaves and roots of NK, spond to the cytoplasmic and vacuolar compartments
even after 24 h, there was no significant decrease in tissue (Zhen et al., 1991). The change in compartmental location
nitrate. When the nitrate remobilization was looked at in of the electrode tip occurred spontaneously without any
more detail using microelectrodes in specific tissues a adjustment of the micromanipulator. Later during the
different pattern emerged. recording (20 min) the N supply was removed but this
To measure the nitrate status of individual cells, double- treatment did not change the nitrate activity reported in
barrelled ion-selective microelectrodes were used in root the cell (Fig. 4). Many measurements were obtained in
 Nitrate storage and remobilization in rice 1733
cytosolic nitrate activity (5.9 mM) was significantly
higher than that in NK (2.9 mM), but there were no
significant differences between the cultivars in the root
cell cytosolic nitrate activities (Fig. 5). Microelectrode
measurements have shown previously that, in barley, the
epidermal layer of root cells was mobilized faster than the
cortical cells during N starvation (van der Leij et al.,
1998). Figure 6 shows nitrate microelectrode measure-
ments of the outer layer of cells in the leaf and root for the

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two cultivars for the 24 h after withdrawal of the external
N supply. Cytosolic and vacuolar measurements were
assigned according to the separation of the two popula-
tions at time t¼0 and the values in Fig. 5. However, for
NK leaf cells there was considerable spread of the data
points, and vacuolar compartmental assignment was
Fig. 2. Tissue dry weight for rice plants grown in hydroponic culture difficult for some of the lower values (Fig. 6C). In YD,
with either 5 mM Ca(NO3)2 or 5 mM ammonium nitrate as N source. leaf cell cytosolic nitrate was maintained at a significantly
These measurements show that both varieties of rice can produce
similar amounts of biomass when grown on high concentrations of higher activity than that measured in other cell types
either N source. Mean values 6SD are shown for a sample size of at during the remobilization period (Fig. 6D). All these
least eight plants. cellular measurements show significant remobilization of
epidermal vacuolar nitrate pools in roots and leaves of
both cultivars. Lines with similar exponential shapes
could be fitted through all the vacuolar nitrate measure-
ments (Fig. 6), and equation parameters were compared
(see Table 1) for the rates of nitrate remobilization in
different tissues and cultivars. The parameter a obtained
from these fits, like the mean vacuolar nitrate activities
(Fig. 5A), reflects the size of vacuolar nitrate pools
(between 40 mM and 55 mM) before starvation. While
Fig. 5 showed no significant differences between cultivars
and tissues before N supply withdrawal, the parameter
a for YD roots was significantly different from the value
for NK leaves (Table 1).
Also the xylem sap nitrate activities of the two rice
cultivars were compared. These measurements showed
significant differences between the two cultivars (Fig. 7A)
with NK showing a higher value even after 24 h N
starvation relative to YD. Comparing the sap volumes
shows that YD produced more over the same time period
but NK delivers a greater total amount of nitrate in the
Fig. 3. The changes in whole tissue nitrate content of rice plants after xylem (Fig. 7B, C). After 24 h of N starvation both
withdrawal of the external N supply. The bar shows changes in the light cultivars showed increased sap volume (Fig. 7B) but the
supply to the leaves, with the dark period shaded (see Materials and
methods for details). Mean values 6SD are shown for a sample size of xylem nitrate content had decreased relatively more in
at least eight plants. YD than in NK (Fig. 7C; by 94% for YD, by 79% for NK).
Two-way ANOVAs comparing the differences between
nitrate content data (Fig. 7C) for the two cultivars were
this way for leaves and roots of the two rice cultivars. highly significant (P <0.01). Also N withdrawal had a
A summary of the mean vacuolar and cytosolic nitrate huge effect on both varieties, but a greater effect on YD
activities in root rhizodermal and leaf epidermal cells is than NK. To compare and confirm this result, xylem sap
shown in Fig. 5. There were no significant differences nitrate was also measured in rice seedlings of the same
between the mean vacuolar nitrate activities of the two age grown hydroponically in 2.5 mM nitrate, and the
cultivars (Fig. 5A). These vacuolar nitrate activities in same pattern of differences between the two cultivars was
epidermal cells are generally higher than those concen- observed (data not shown).
trations measured in the whole tissue (comparing Figs 3 The expression pattern of NR and the activity of the
and 5A). In contrast, for leaf cells, in YD the mean enzyme was measured and compared in both cultivars
1734 Fan et al.

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Fig. 4. Nitrate-selective microelectrode recordings obtained from a YD rice root rhizodermal cell. The YD rice plant was cultivated in 10 mM nitrate
as N source. The electrode tip initially records a compartmental concentration of about 65 mM and a membrane potential of
94 mV, but the
recording shifts to a smaller value of about 2.5 mM nitrate with a membrane potential of
110 mV after 7 min. These two nitrate activities are
believed to correspond to the vacuole and cytosol, respectively (Zhen et al., 1991). After 15 min in the nitrate bathing solution the roots were
changed from 10 mM to 0 mM nitrate during the recording. The hatched bar on the figure shows when the root was immersed in 10 mM nitrate and
the open bar when it was immersed in 0 mM nitrate. The arrow represents 5 min and each tick on the x-axis is a 5 min interval.

before and after 24 h of N starvation. There was decreased As leaf tissue nitrate pools did not significantly decrease
expression of OsNia1 and OsNia2 in both YD and NK in either cultivar 24 h after the withdrawal of N supply,
leaves after N starvation (Fig. 8A, B). In rice, like barley stem nitrate pools were also compared (see Fig. 9).
(Sueyoshi et al., 1995), the transcript levels of both genes Statistical analysis of these tissue measurements showed
were decreased, but for OsNia1 the effect was greater than a significantly higher amount (Fig. 9A) and concentration
for OsNia2 (Fig. 8A, B). Pooling the results in Fig. 8B, C (Fig. 9B) present in YD stems when compared with NK
for both cultivars showed there was only a strong before removal of the N supply to the roots. The amount
correlation between OsNia1 transcript and NRAact of stem nitrate in YD and NK decreased significantly after
(r¼0.97, P <0.05). The activity of NR is regulated by 24 h of N withdrawal (Fig. 9A), but only NK showed a
reversible Mg-dependent phosphorylation, which has been significant decrease in stem nitrate concentration (Fig. 9B).
shown to be responsible for the light-regulated fluctua- These results suggest that there may be a difference be-
tions observed in its activity in leaf tissue (Tucker et al., tween cultivars in the stem water content that is associated
2004). Phosphorylation enables a 14-3-3 inhibitory pro- with the remobilization of vacuolar stored nitrate. How-
tein to interact with the NR enzyme and suppress its ever, the percentage water content was not significantly
activity (reviewed by Kaiser and Huber, 2001; Kaiser different between cultivars (data not shown).
et al., 2002). Consequently, in the presence of MgCl2,
only the NR that is active in vivo is measured, whereas in
the absence of MgCl2 a measurement of the total activity
Discussion
is obtained. Before N starvation the total NRA (NRAmax,
NRA-Mg2+ maximum) was similar in both YD and NK Nitrogen use efficiency is relatively low in a flooded rice
leaves (Fig. 8C). After 24 h N starvation NRAmax had crop because of N losses through denitrification, ammonia
significantly decreased by 80% in NK but not YD volatilization, run-off, and leaching (Vlek and Byres,
(P <0.01). A different pattern was observed for NRAact 1986). Previous work has reported considerable variation
(NRA+Mg2+ active) in the two cultivars. Before N in N utilization by field-grown rice, and this occurs for
starvation NRAact was significantly greater in YD than both grain and biomass production (Koutroubas and
NK leaves. In fact NRAact and NRAmax were not Ntanos, 2003, and references therein). It has been
significantly different in YD but only 27% NR was active demonstrated in the present study that these two Chinese
in NK leaves. After 24 h N starvation, NRAact was rice cultivars show significantly different levels of NUE
decreased in both YD and NK leaves. Following N for biomass production in both hydroponic and soil
withdrawal 44% NR was in the active form in YD while culture (Fig. 1). Biomass production was very similar
29% of NR was active in NK leaves (see Fig. 8C). under mixed N or nitrate only supply for both cultivars
 Nitrate storage and remobilization in rice 1735
indica rice. In field experiments, when several different
cultivars of japonica and indica rice were compared, the
results showed no significant differences in NUE between
the two types of rice for both grain yield and biomass
production (Koutroubas and Ntanos, 2003).
Cellular nitrate pools
Whole tissue nitrate gives a measure of cortical (root) and
mesophyll (leaf) cell vacuolar concentrations (Zhen et al.,

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1991; Cookson et al., 2005). Microelectrode measurements
in epidermal cells gave higher vacuolar activities than
those values measured in whole tissue, which is domi-
nated by the cortical and mesophyll cells (comparing Figs
3 and 5A). Taken together these data suggest that nitrate is
preferentially stored in the vacuoles of rice epidermal cells.
Single cell micro-sampling methods in barley leaves have
previously shown that nitrate was preferentially stored in
the upper epidermis, and this pattern was independent of
light supply and development (Fricke et al., 1994). These
results suggest that nitrate accumulation in the upper leaf
epidermis may be a more general feature of cereal crops.
Measuring whole tissue nitrate changes after 24 h of N
starvation provided some evidence that the cultivar YD
depleted root vacuolar nitrate pools faster than NK (Fig.
3), but more data beyond 24 h starvation are needed to
check if this trend continues. As YD had better NUE than
NK, the accessibility of vacuolar nitrate pools may be
worthy of more investigation. In the epidermal layer of
barley root cells, vacuolar stored nitrate was mobilized
faster than that in the cortical cells (van der Leij et al.,
Fig. 5. The mean vacuolar (A) and cytosolic (B) nitrate activities in 1998), and in these roots the time course for remobiliza-
rhizodermal cells of YD and NK roots measured with nitrate-selective tion of nitrate stored in cortical cell vacuoles was similar
microelectrodes. Rice plants were previously cultivated in 10 mM ni- to that measured in the whole roots. Microelectrode mea-
trate as N source for 2 weeks. Each data point shows the mean 6SD of
six repetitions. Two-way ANOVA showed that YD leaf cytosolic nitrate surements of vacuolar and cytosolic nitrate pools in the
was significantly different from the other cytosolic data (P <0.01). outer cell layers of leaves and roots showed significant
differences in the rate of nitrate remobilization (b in Table
1) only between leaves and roots in YD (Fig. 6B, D) but
(Fig. 2), but YD consistently showed better NUE (Fig. 1). not in NK or between cultivars (Fig. 6A, C). Interestingly,
This effect on biomass production for these cultivars at both before and after N supply withdrawal the steady-
lower N supply has been reported previously (Fan et al., state cytosolic nitrate activity of YD leaf epidermal cells
2005). The hypothesis that differences in the ability to was significantly higher than that measured in equivalent
remobilize stored nitrate may help explain the improved cells of NK (Figs 5 and 6). These differences between leaf
NUE shown by YD when judged against NK was tested. cells are not related to photosynthetic activity of the cells
The higher grain-yielding japonica rice NK (Fan et al., (Cookson et al., 2005) as epidermal cells of both cultivars
2005) showed poorer NUE for biomass production when have no green plastids. Furthermore, the cytosolic nitrate
compared with the indica rice YD. NUE for grain yield activities of both cultivars are not significantly different in
in these two cultivars has not been measured and this root cells. As YD has better NUE than NK, a higher leaf
parameter has more obvious practical value for crops but epidermal cytosolic nitrate activity (Fig. 5B) may contrib-
it must subsume the term for biomass. Here the focus has ute to this trait in rice and may result from a different
been on biomass NUE because nitrate pools are more tissue distribution of NRA in leaves of the two cultivars
likely to be important for vegetative growth, and environ- (Cookson et al., 2005).
mentally damaging leaching losses occur during this de-
velopmental stage. The difference in biomass NUE Nitrate reductase activity
reported here probably reflects a distinction between these The vacuole is the site of nitrate storage in cells (Miller
cultivars and is not a general feature of japonica and and Smith, 1996; van der Leij et al., 1998; Kronzucker
1736 Fan et al.

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Fig. 6. The nitrate activities in epidermal cells of rice roots and leaves measured with ion-selective microelectrodes during the first 24 h after removal
of the external nitrate supply: (A) NK roots; (B) YD roots; (C) NK leaves; (D)YD leaves. The YD rice plants were cultivated in 10 mM nitrate and
then nitrate was removed (no nitrogen source) from the cultivation solution. The nutrient solution for all these double-barrelled nitrate-selective
microelectrode measurements contained no N. Lines were fitted through the vacuolar data using an exponential relationship y¼ae
bx. The parameters
for these fits are given in Table 1.

Table 1. The parameters obtained from the vacuolar remobil- nitrate translocation into xylem. Barley roots have measur-
ization curves fitted using the data in Fig. 6 and showing values able NRA (van der Leij et al., 1998) but in the root
for different cultivars and tissues epidermal cells of both species the removal of the external
nitrate supply has no short-term effect on cytosolic nitrate
Cultivar Tissue Parameters of fit y¼ae
bx
activity. These results suggest that the vacuole restores
a b cytosolic nitrate activity in these cells and this is demon-
strated by the decrease in this storage pool (remobiliza-
NK Root 48.963.2 0.02960.006
Leaf 41.164.9 0.03860.012 tion) shown in Fig. 6.
YD Root 52.662.4 0.03860.005 In the leaf, NRA may be an important factor de-
Leaf 50.562.7 0.05160.007 termining nitrate utilization by the rice plants. In both
cultivars, withdrawal of N supply has decreased both nia
transcripts and NRAact (Fig. 8A–C), but YD consistently
et al., 2000; Miller et al., 2001). In plant cells several maintained a higher proportion of NRAact before and after
different processes contribute to establish an equilibrium N starvation (Fig. 8C). The higher cytosolic nitrate ac-
that determines the size of cytosolic and vacuolar nitrate tivity (Fig. 5B) and faster rates of leaf vacuolar nitrate
pools, and the importance of these can be different in leaf remobilization (Fig. 6; Table 1) in YD may be important
and root cells. For example, in the Arabidopsis leaf factors for maintaining NRAact and OsNia expression in
mesophyll cell, cytosolic nitrate activity was very de- this cultivar. In barley, the expression of both NADH and
pendent on NRA (Cookson et al., 2005). In contrast, in NAD(P)H NRA was suggested to be dependent on nitrate
a rice root, as NRA was not detectable the cytosolic flux (Sueyoshi et al., 1995). Alternatively, the greater
nitrate pool was maintained by nitrate uptake across the NRA in YD, relative to NK, will provide a stronger sink
plasma membrane, transport across the vacuole, and the pulling nitrate from the epidermal cell vacuoles and xylem
 Nitrate storage and remobilization in rice 1737

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Fig. 7. The response of xylem sap to the withdrawal of external N supply. (A) The nitrate activity of the xylem sap samples was measured using
nitrate-selective microelectrodes. (B) The volume of xylem sap samples collected was measured and the mean 6SD is shown. (C) The total amount
of nitrate in each sap sample. YD and NK were cultivated in 10 mM nitrate and xylem sap samples were collected before and after 24 h of N
starvation.

sap (Fig. 6D). Previous work studying the cause of diurnal Xylem sap nitrate
changes in N metabolism of tobacco leaves suggested that The fact that the xylem sap nitrate concentration was
an imbalance between nitrate reduction, uptake, and higher in NK when compared with YD (Fig. 7A) might
ammonium metabolism early in the light period gave rise suggest a greater net transfer from root to shoot in NK.
to the changes in leaf metabolite pools including nitrate The nitrate concentration of xylem sap is often used as an
(Matt et al., 2001). Matt et al. (2001) suggested that the indicator of the N status of a plant as there is good
ability to increase NRA rapidly might be a selective evidence that this parameter, like NRA, increases in a way
advantage in a less-favourable environment. An ability to that is directly dependent on the concentration of the
maintain nitrate assimilation during the withdrawal of N external supply (Andrews, 1986). N starvation decreased
supply might be important for NUE. The fact that YD the nitrate concentration of the xylem sap of both culti-
maintained higher NRAact even after N starvation pro- vars (Fig. 7A). During the first 24 h of N supply
vides support for this idea. withdrawal there was some evidence that the roots of
1738 Fan et al.

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Fig. 8. Changes in leaf nitrate reductase expression and activity during N supply withdrawal: (A) expression pattern for nia1 and nia2;
(B) quantification of the expression pattern for nia1 and nia2 relative to actin, before and after N supply withdrawal; (C) nitrate reductase activity
NRAact and NRAmax in leaves of YD and NK rice cultivars during nitrate depletion. Mean values 6SD are shown, four replicates of each. Rice plants
were cultivated in 10 mM nitrate and then N starved for 24 h.

YD remobilized more nitrate than NK (Fig. 3). The show differing relationships between nitrate contents of
measurements of xylem sap nitrate content during this N the xylem sap and stem—NK has more nitrate in the
starvation period provide evidence for a larger relative xylem and less accumulation in the stem while YD shows
decrease in delivery of nitrate to the shoot in YD when the opposite pattern. These results suggest that there is
compared with NK (Fig. 7C). However, NK under both little value in xylem sap analysis for comparing crop N
conditions was actually supplying more nitrate to the status between cultivars. The different responses of stem
shoot than YD even after 24 h of N starvation. So where and xylem sap nitrate during short-term N starvation
is this nitrate coming from as the external supply has been shown by the two cultivars may suggest important
removed and there is no evidence of a significant decrease relationships between these parameters and NUE that are
in leaf tissue nitrate for NK during this time? Leaf tissue worthy of further investigation.
nitrate measurements were made at the middle of a 4 cm Rice shows a slower decrease in vacuolar and xylem
section of the emerged leaf, as the stem store, including nitrate during N starvation when compared with barley
the younger parts of the leaf, is a possible source. (Sueyoshi et al., 1995; van der Leij et al., 1998).
Analysis of stem tissue showed that there was signifi- Nonetheless, like barley, both rice cultivars showed
cantly more nitrate stored in this tissue in YD when a pattern of vacuolar nitrate remobilization to maintain
compared with NK (Fig. 9) but both cultivars remobilize the nitrate activity in the cytoplasm (Figs 3 and 6), but,
this nitrate source after 24 h of N starvation. This stem in contrast to barley, less efflux of cytosolic nitrate into
portion of the plant provides nitrate that maintains xylem xylem when N starved. The fact that the lower NUE
delivery to the leaf in both cultivars. The two cultivars cultivar has higher xylem sap nitrate might suggest that
 Nitrate storage and remobilization in rice 1739
Conclusions
Among Chinese rice cultivars there appears to be great
scope for improving NUE (Peng et al., 2006). The two
Chinese rice cultivars chosen for this study showed
consistently significant differences in NUE for biomass
accumulation in soil and hydroponics with both nitrate
and mixed N supplies. Cellular nitrate pools are important
for both tissue expansion and N storage during vegetative

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growth (McIntyre, 1997). This developmental stage is
when the most environmentally damaging leaching losses
from the crop occur (Peng et al., 2006). A comparison of
nitrate physiology during the withdrawal of N supply was
used to identify some differences between the two
cultivars that are likely to be important for NUE. These
cultivar differences for rice can be listed as follows.

(i) Leaf epidermal cell cytosolic nitrate has a higher

steady-state value and the ability to maintain NRAact
during 24 h of N deprivation. These parameters may
be linked as NRAact may depend on the cytosolic
nitrate activity.
(ii) Stem-stored nitrate provides an important supply for
remobilization to support vegetative growth. Stem,
rather than leaf, tissue nitrate measurements are likely
to be an earlier measure of changes in crop N status.
An ability to remobilize root cortex vacuolar-stored
nitrate during N starvation may be important, al-
though this occurs later than stem nitrate pools.
(iii) An ability to store more nitrate in the stem can indi-
cate lower concentrations in the xylem sap nitrate
that may result in an optimization of supply to the
leaf for better NUE. Xylem sap analysis is not
appropriate for comparing crop N status or NUE
between cultivars.

These points summarize some traits for comparing NUE

between plants of the same species but may also be of
more general relevance to all cereal crops.

Acknowledgements
This work was funded in China by the National Natural Science
Foundation (grant number 30390082), the National Basic Research
Program 973 (grant No. 2005CB120903), and a grant from Jiangsu
Province (BK2004102). Rothamsted Research is grant-aided by the
Fig. 9. Changes in stem nitrate after 24 h of N supply withdrawal: Biotechnology and Biological Sciences Research Council (BBSRC)
(A) amount of stem nitrate per plant; (B) stem nitrate concentration of the UK. The authors wish to thank Stephen Powers at Roth-
(lmol g
1 FW). Rice plants were cultivated in 10 mM nitrate and then
N starved for 24 h. Mean values 6SD are shown for a sample size of at amsted for statistical advice.
least eight plants.
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