Plant Soil (2015) 386:65–76
DOI 10.1007/s11104-014-2240-4
REGULAR ARTICLE
Root plasticity and its functional roles were triggered by water
deficit but not by the resulting changes in the forms of soil N
in rice
Thiem Thi Tran & Mana Kano-Nakata &
Roel Rodriguez Suralta & Daniel Menge &
Shiro Mitsuya & Yoshiaki Inukai & Akira Yamauchi
Received: 6 January 2014 / Accepted: 12 August 2014 / Published online: 29 August 2014
# Springer International Publishing Switzerland 2014
Abstract
Background The functional roles of root plasticity in
rice adaptation to drought conditions may vary with soil
nitrogen (N) conditions.
Aims To examine if: promoted root system plastic development triggered by mild drought stress and enhanced by N application would contribute to the increase in soil water uptake, and if expression of root
system plasticity would be affected by different forms of
N applied into the soil.
Methods Chromosome segment substitution line
(CSSL) 50 and Nipponbare genotypes were grown
Responsible Editor: Tim Simon George.
Electronic supplementary material The online version of this
article (doi:10.1007/s11104-014-2240-4) contains supplementary
material, which is available to authorized users.
T. T. Tran : R. R. Suralta : D. Menge : S. Mitsuya :
A. Yamauchi (*)
Graduate School of Bioagricultural Sciences, Nagoya
University, Chikusa, Nagoya 464-8601, Japan
e-mail: [email protected]
T. T. Tran
Faculty of Agronomy, Vietnam National University of
Agriculture, GialamHanoi, Vietnam
M. Kano-Nakata : Y. Inukai
International Cooperation Center for Agricultural Education,
Nagoya University, Chikusa, Nagoya 464-8601, Japan
R. R. Suralta
Agronomy, Soils and Plant Physiology Division, Philippine
Rice Research Institute (PhilRice), Maligaya, Science City of
Muñoz, Nueva Ecija 3119, Philippines
under continuously waterlogged (CWL) and water deficit (WD) conditions. In rootbox (25 cm×40 cm×2 cm)
experiment, three fertilizer N levels; (30 (low), 60
(standard) and 120 mg N (high) per rootbox) were used
while in pot (5 L) experiment, six N forms (NH4+-N
alone, NO3−-N alone, combined NH4+-N and NO3−-N
with and without dicyandiamide (nitrification inhibitor)
were used at the rate of 360 mg N per pot.
Results In both experiments, CSSL50 and Nipponbare
had no significant differences in shoot and root growth
regardless of N levels and N forms under CWL conditions. However, under WD conditions, CSSL50 had
significantly greater dry matter production (DMP) than
Nipponbare due to the greater ability of the former for
maintaining soil water uptake and photosynthesis. The
observed higher water uptake and photosynthesis in
CSSL50 under WD was closely related to its promoted
root system development due to plasticity, which were
significantly greater at high N than at low N level. The
extent of promotion in root system development based
total root length was not significantly different among N
forms.
Conclusions The root system plasticity of CSSL50 in
response to WD was expressed at a greater degree with
high level of N applied and the functional roles of root
plasticity for greater soil water uptake and DMP were
due to WD regardless of N forms.
Keywords Ammonium . Chromosome segment
substitution lines . Dry matter production . Nitrate .
Root plasticity . Water deficit
66
Abbreviations
CSSLs chromosome segment substitution lines
DAS
days after sowing
SMC
soil moisture content
CWL
continuously waterlogged
WD
water deficit
N
nitrogen
G
genotype
DMP
dry matter production
Introduction
Water stress is one of the most important environmental
limitations on the productivity of rice in rainfed system.
In the upland systems, soils are mostly in aerobic state
throughout the cropping period. In rainfed lowland and
flood-prone ecosystems, waterlogging and submergence may occur. These changes in soil moisture have
marked effects on the soil conditions, availability of
nutrients and water, and root system development and
functions (Kondo et al. 2005; Kato et al. 2007).
Nitrogen (N) fertilizer application is important for
root system development and functions. Nitrogen fertilization enhanced the production of fine roots and root
hairs (Fageria 2010), which results in an increase in root
dry weight, total root length, root length density and root
diameter (Fageria 2010) and thus increase the efficiency
of water and N uptake (Suralta 2010). In addition, N
application can increase grain yield of rice under rainfed
lowland conditions with occasional exposure to water
deficit (Wade et al. 1999; Castillo et al. 2006) and mild
water stress in water saving system (Belder et al. 2005).
The interaction between water and nitrogen is critical in
rice adaptation to stress-prone environments such as
rainfed lowland ecosystems where both water and nutrient are limited, and thus affect root functions (Suralta
2010; Tran et al. 2014), photosynthesis and transpiration
(Otoo et al. 1989), and dry matter production (Allahyar
2011).
Roots play an important role in the adaptation to
drought conditions in rice (Yamauchi et al. 1996)
through the development of deep and extensive root
systems (Serraj et al. 2004), which includes thick roots
(Price et al. 2000) and increased root length density
(Siopongco et al. 2005) as a result of the plasticity in
lateral root development (Bañoc et al. 2000; Kamoshita
et al. 2000). These adaptations are associated with
Plant Soil (2015) 386:65–76
increased water extractions (Kamoshita et al. 2000,
2004; Siopongco et al. 2005, 2006; Kato et al. 2007),
and nutrient uptake (Suralta 2010). As such, the root
plasticity that is expressed in response to such heterogeneous soil environment is one of the key traits for better
plant adaptation (Yamauchi et al. 1996; Wang and
Yamauchi 2006). Most of the genetic variations in root
system architectures are controlled by a suite of quantitative trait loci (QTL) (Dorlodot et al. 2007). QTLs
associated with root trait responses to varying soil water
regimes and/or nutrient status affecting root system
architecture (RSA) have been targeted in many cereal
species such as in rice (Obara et al. 2010; Uga et al.
2011), maize (Landi et al. 2010; Ruta et al. 2010), wheat
(Laperche et al. 2006; Sharma et al. 2011), barley (Nevo
and Chen 2010) and pear millet (Yadav et al. 2011).
Previously we precisely quantified and analyzed the
functional roles of root plasticity in plant adaptations
using genetically related lines such as the chromosome
segment substitution lines (CSSLs), a mapping population in which each line carries a single or a few defined
chromosome segment of donor genome and has a pure
genetic background from a recurrent genotype (Kano
et al. 2011; Kano-Nakata et al. 2011; Niones et al. 2012;
2013; Tran et al. 2014) and introgression lines (KanoNakata et al. 2013). Using CSSLs from a cross between
the recurrent parent Nipponbare (japonica type) and the
donor parent Kasalath (indica type), a selected CSSL50
out of the 54 CSSLs was examined and compared with
the recurrent parent Nipponbare under various intensities of drought in the field (Kano et al. 2011) and
greenhouse (rootbox) conditions (Kano-Nakata et al.
2011). In these studies, CSSL50 consistently showed a
significantly greater plasticity in root system development based on total root length than Nipponbare due to
promoted lateral root branching and elongation under
mild drought stress conditions. Furthermore, the expression of such root plasticity was greater at high N than at
low N level, which greatly contributed to greater dry
matter production (Tran et al. 2014) probably due to the
enhanced soil water uptake which was not quantified
because these experiments were conducted in the field
where direct and precise measurements of root water
uptake is difficult. It is not known, however, whether the
root plasticity exhibited by CSSL50 under mild drought
stress was solely due to the effect of moisture deficit or
to the interactions between the soil moisture conditions
and parallel changes in N form. The availability of N in
the soil depends on soil moisture conditions. In the
Plant Soil (2015) 386:65–76
paddy field, ammonium (NH4+) rather than nitrate
(NO3−) is a major source of N for rice while under
aerobic field conditions, NH4+-N is rapidly converted
to NO3− and/or a mixture of NH4+ and NO3− (Qian et al.
2004). Both changes in N supply forms and water status
under non-flooded soil conditions influenced the growth
of rice plants (Guo et al. 2007). Also, the application of
nitrate as N source increased total root length and root
volume in rice compared with ammonium nutrition
(Gao et al. 2010). Thus, we hypothesized that root
developmental plasticity triggered by drought stress
may be influenced not only by the amount of available
N in the soil but also by N forms.
This study, examined if promoted development of
root system due to the plasticity triggered by mild
drought stress and enhanced by optimal amount of N
application would contribute to increased water uptake,
and the expression of such plasticity in root system
development would be affected by different forms of
N applied into the soil. This study was conducted in pots
and rootboxes to precisely quantify the expression of
root plasticity and its roles in water uptake, which are
easier to achieve than under field experiment.
Materials and Methods
Plant materials
CSSL50 derived from Nipponbare/Kasalath cross, and
the recurrent parent Nipponbare, were used. The seeds
were supplied by the Rice Genome Research Center of
the National Institute of Agrobiological Sciences, Japan
(http://www.rgrc.dna.affrc.go.jp/ineNKCSSL54.html).
CSSL50 was selected because of its greater plasticity in
root system development based on total root length
through promoted lateral root branching and
elongation compared with Nipponbare under mild
drought stress conditions (Kano et al. 2011; KanoNakata et al. 2011; Tran et al. 2014). The experiments
were conducted in a vinyl house at the experimental
field of Nagoya University, Japan (136°56′6′′ E, 35° 9′
5′′ N).
Experiment 1 (rootbox)
The seeds of each genotype were soaked in water mixed
with fungicide (benomyl (benlate), 0.15 % w/v) and
incubated in a seed germinator maintained at 28 °C for
67
72 h prior to sowing. Three pre-germinated seeds were
sown in a PVC rootbox (25 cm×2 cm×40 cm, L×W×
H) filled with 2.5 kg of air-dried sandy loam soil following the method of Kano-Nakata et al. (2012). The
seedlings were thinned to one seedling per box at 3 days
after sowing (DAS).
Soil moisture treatments, continuously waterlogged
(CWL-control); and two water deficit (WD) conditions:
25 % w/w (−0.014 MPa) and 20 % w/w (−0.040 MPa),
similar to those of Kano-Nakata et al. (2011) were used.
In CWL, water level was maintained at 2 cm above the
soil surface from 5 DAS until the end of the experiment
at 38 DAS. In WD conditions, each rootbox was
weighed daily and the amount of water lost was
replenished and recorded as evapotranspiration. Four
rootboxes without plants were also prepared to measure
the amount of water lost through evaporation. The
whole plant transpiration was estimated as the difference
in water loss between rootboxes with and without
plants. The water use was calculated as the accumulated
daily whole plant transpiration from 7 DAS up to the
termination of the experiment (38 DAS).
The standard N fertilizer level was applied following
those of Suralta et al. (2010) and Kano-Nakata et al.
(2011). The N fertilizer (urea: 46%N) was used at three
different levels: 30 mg (low), 60 mg (standard) and
120 mg N (high). Each level of N fertilizer was thoroughly mixed with 80 mg phosphorus (single superphosphate: 17.5 % P2O5) and 70 mg potassium (KCl:
60 % K2O) into the 2.5 kg air-dried soil per rootbox
prior to seed sowing.
Sampling was done at 38 DAS. Four rootboxes (1
rootbox=1 replication) were harvested for each treatment combination. The shoots were cut at the stem base
and oven-dried at 70 °C for 3 days prior to recording of
the dry weight. The roots were sampled using a
pinboard and transparent perforated plastic sheet following the method of Kano-Nakata et al. (2012).
Soil samples were also taken at 38 DAS. From each
rootbox, the soil was sampled from three depths: 0–5,
17.5–22.5 and 35–40 cm below the soil surface. The soil
samples were separately air-dried and sieved on a 2 mmmesh screen. For ammonium analysis, soil samples
were extracted with KCl solution (5 g of soil in 50 ml
of 2 M KCl), shaken for 60 min, and filtrated through a
0.22 mm thick filter paper (Advantec Grade No. 5C).
The concentration of NH4+-N was determined using the
indophenol-blue method (Weatherburn 1967). For nitrate analysis, soil samples were extracted with distilled
68
water (5 g of soil in 100 ml of distilled water), shaken for
30 min, and filtrated first through filter paper (Advantec
Grade No. 5C) and then again through microfilter
(0.45 μm thickness). The concentration of NO3−-N
was measured using an ion chromatography system
(Shim-pack IC-A3; Shimadzu, Kyoto, Japan) and
expressed in mg N kg−1 soil.
The collected root samples embedded between the
plastic sheets were washed free of soil in running water.
Cleaned root samples were stored in FAA (formalin:
acetic acid: 70 % ethanol in 1:1:18 ratio by volume)
solution for preservation and further measurements of
various root components. The length of nodal roots was
measured using a ruler and the total number of nodal
roots at the base of each stem was manually counted.
For total root length measurements, roots were spread
on the transparent sheets with minimal overlapping.
Digital images were then taken using an Epson scanner
(ES2200) at 300 dpi resolution. The total length of each
root sample was analyzed using WinRHIZO software v.
2007d (Regent Instruments, Quebec, Canada).
Plant Soil (2015) 386:65–76
were measured using a portable photosynthesis analyzer
(LI-6400, LI-COR, Lincoln, NE, USA) on the abaxial
side of the topmost fully-developed leaf of the main
stem between 9 AM and 11 AM at 40 DAS using the
following system settings: leaf temperature, 30 °C; CO2
concentration, 380 μ L L−1; relative humidity, 65–75 %;
quantum flux density, 1200 μmol m−2 s−1.
Plant and soil samplings were done at 50 DAS. Four
pots (1 pot=1 replication) were harvested for each treatment combination. The shoot and root growth parameters were measured in similar way to those in
Experiment 1.
For soil sampling, the soil profile in each pot was
divided into three depths: 0–5, 7.5–12.5 and 15–20 cm
from the soil surface. The soil samples were collected
from three random points from each depth. Each soil
sample was separately air-dried and sieved on a 2 mmmesh screen. The concentration of NH4+-N and NO3−-N
were analyzed similar to that of the Experiment 1.
Statistical analysis
Experiment 2 (pot)
To examine whether the expression of the root plasticity
of CSSL50 in response to mild water deficit is affected
by N form, plastic pots (20 cm in height and 16 cm in
diameter) filled with 4.0 kg of air-dried sandy loam soil
were used for the experiment. The same set of water
treatments, such as CWL and WD condition at 20 % w/
w as described in the rootbox experiment (Experiment
1) was used in this experiment. Six N form treatments at
the same rate used by Yamauchi et al. (1988) at
360 mg N per pot were prepared as follows: NH4+-N
alone (A); NH4+-N with nitrification inhibitor (ADCD);
NO3−-N alone (N); NO3−-N with nitrification inhibitor
(NDCD); combined NH4+-N and NO3−-N (AN); combined NH4+-N and NO3−-N with nitrification inhibitor
(ANDCD). The source of NH4+-N was (NH4)2SO4,
while that of NO3−-N was Ca(NO3)2. The nitrification
inhibitor, dicyandiamide (DCD) (C2H4N4), was applied
at the rate of 100 mg pot−1. The P from KH2PO4, and
K from KCl were also added based on the rate
used by Yamauchi et al. (1988) at 480 mg and
420 mg pot−1, respectively. Each treatment was
thoroughly mixed into the soil of each pot before seed
sowing.
The water uptake measurement was performed similar to that in Experiment 1. The photosynthetic rates
Both experiments were arranged in split-split plot design in RCBD with four replications. The soil moisture
treatments (Experiment 1 and Experiment 2) was
assigned to the mainplot while either N levels
(Experiment 1) or N form (Experiment 2) treatments
was assigned in the subplot. Furthermore, the genotypes, which were common in both experiments, were
assigned in the sub-subplots. The analysis of variance
(ANOVA) for main effects of SMC, N and G and their
two-way and three-way interactions were generated
using CropStat version 7.2 (IRRI, 2009). The difference
in average values among genotypes was tested using the
least significant difference (LSD) at 5 % level of
significance.
Results
NH4+-N and NO3−-N in soil
The amount of NH4+-N and NO3−-N in soil were not
significantly different among three depths as well as
between CSSL50 and Nipponbare regardless of soil
depth in both experiments (data not shown). Thus, the
values presented herein are the average of three soil
depths of the two genotypes (Tables 1 and 2). Table 1
Plant Soil (2015) 386:65–76
69
Table 1 Effect of N application levels on the amount of NH4+-N
(mg N kg soil -1) and NO3−-N (mg N kg soil -1) in the soil under
continuously waterlogged (CWL) and water deficit (WD) conditions at 25 and 20 % w/w of SMC (Experiment 1)
SMC
NH4+-N
NO3−-N
N level
(mg N rootbox−1) (mg N kg soil−1) (mg N soil kg−1)
CWL
30
6.7a
60
11.4b
0
120
15.5c
0
30
3.0a
43.7a
60
7.5b
66.2a
25 %
20 %
0
120
9.5c
89.2b
30
3.6a
49.0a
60
7.1b
77.7b
120
8.7c
90.5c
The source of N applied was urea fertilizer (46-0-0 NPK)
Values followed by the same letter in a column within each soil
moisture treatment are not significantly different at the 5 % level
shows the effect of N application levels on the amount
of NH4+-N and NO3−-N in soil under CWL and WD
conditions at 38 DAS in Experiment 1. The result
showed that the increase in N application increased the
amount of NH4+-N in soil regardless of soil moisture
treatments. On the other hand, the amount of NO3−-N in
soil increased as N application increased under WD
conditions (Table 1). Under CWL, NO3−-N in soil was
negligible regardless of N levels (Table 1) and N forms
(Table 2) applied. Under saturated and waterlogged soil
conditions, the denitrification process is favored by lack
of oxygen, which resulted in four to six-fold higher rates
of denitrification than the aerobic upland soils (Aulakh
et al. 2000). The nitrification inhibitor (i.e. ADCD)
under waterlogged soils was rendered non-functional
because of the limitation of O2. As a result, the anticipated N form was NH4+.
In both CWL and WD (20 % w/w) conditions, the
amount of NH4+-N in soil was higher at ADCD and
ANDCD than at A and AN treatments (Table 2,
Experiment 2). In contrast, the amount of NO3−-N in
soil was lower at ADCD and ANDCD than at A and AN
treatments under WD (20 % w/w) conditions only
(Table 2).
Experiment 1 (rootbox)
NH4+-N
Table 2 Effect of N forms on the amount of
(mg N kg
soil -1) and NO3−-N (mg N kg−1 soil) in soil under continuously
waterlogged (CWL) and water deficit (WD) conditions at 20 % w/
w of SMC (Experiment 2)
SMC
CWL
20 %
NH4+-N
(mg N kg−1 soil)
NO3−-N
(mg N kg−1 soil)
A
13.2c
0.0
ADCD
38.9e
0.0
N
0.0a
0.0
NDCD
0.0a
0.0
AN
9.4b
0.0
ANDCD
19.3d
0.0
A
2.2c
60.1a
N form
ADCD
7.1d
47.5a
N
0.0a
160.3c
NDCD
0.0a
159.0c
AN
0.8b
103.0b
ANDCD
1.3b
93.8b
A, NH4+ -N alone; ADCD, NH4+ -N with dicyandiamide; N,
NO3− -N alone; NDCD, NO3− -N with dicyandiamide; AN, combined NH4+ -N and NO3− -N; ANDCD, combined NH4+ -N and
NO3− -N with dicyandiamide
Values followed by the same letter in a column within each soil
moisture treatment are not significantly different at the 5 % level
Responses in shoot and root growth, and water use
to water deficit as affected by different levels of N
application
The shoot dry weight, water use and total root length
under CWL and WD (25 % and 20 % SMC w/w)
conditions applied with three levels of N are shown in
Table 3. The ANOVA showed significant effects of soil
moisture content (SMC), nitrogen (N) level and genotype (G), and the interaction between SMC x G on shoot
dry weight, water use and total root length. Furthermore,
the SMC x N level interaction was significant for shoot
dry weight, water use and total root length. On the other
hand, the N level x G and SMC x N level x G interactions were not significant for all of the traits examined.
Under CWL, the shoot dry weight, water use and
total root length was not significantly different between
CSSL50 and Nipponbare regardless of N application
levels (Table 3). In contrast, under WD conditions, shoot
dry weight, water use and total root length significantly
differed between CSSL50 and Nipponbare but the magnitude of differences varied with SMC and N levels.
Furthermore, the water use efficiency was not significantly different between the two genotypes regardless of
70
Plant Soil (2015) 386:65–76
Table 3 Shoot dry weight (SDW, mg plant−1), water use (WU, g
plant−1 day−1) and total root length (TRL, cm plant−1) of
Nipponbare and CSSL50 grown under continuously waterlogged
(CWL) and water deficit (WD) conditions at 25 and 20 % w/w of
SMC as affected by three N application levels (Experiment 1)
Soil moisture
content
N level
(mg N rootbox−1)
Genotype
SDW
(mg plant−1)
CCSL50/
Nipponbare
WU
(g plant−1 day−1)
CCSL50/
Nipponbare
TRL
(cm plant−1)
CCSL50/
Nipponbare
CWL
30
Nipponbare
1090.3a
1.036
20.6a
1.009
4778.4a
0.992
60
120
25 % w/w
30
60
120
20 % w/w
30
60
120
CSSL50
1129.8a
Nipponbare
1293.5a
CSSL50
1245.0a
Nipponbare
1463.8a
CSSL50
1450.5a
Nipponbare
932.5a
CSSL50
1029.5a
Nipponbare
1063.0b
CSSL50
1207.3a
Nipponbare
994.0b
CSSL50
1101.8a
Nipponbare
884.3b
CSSL50
948.5a
Nipponbare
995.8b
CSSL50
1115.8a
Nipponbare
927.5b
CSSL50
1022.0a
20.8a
0.963
22.5a
4739.0a
0.979
22.0a
0.991
23.9a
10.6b
0.986
11.1b
1.135
11.3b
1.229
8.2b
1.163
8.6b
8.3b
11.2a
4168.1b
1.159
4195.5b
1.100
3474.3b
1.110
3855.7a
1.465
12.6a
1.102
1.077
4616.4a
1.246
10.2a
1.121
3979.0b
4832.9a
13.1a
1.073
0.982
4284.8a
13.6a
1.108
5787.8a
5685.2a
12.0a
1.136
0.981
4844.6a
23.5a
1.104
4938.4a
3716.5b
1.135
4218.9a
1.346
3621.6b
1.129
4088.2a
SMC
***
***
***
N level
***
***
***
G
**
***
**
SMC x N level
***
*
**
SMC x G
*
***
*
G x N level
ns
ns
ns
SMC x N level x G
ns
ns
ns
SMC soil moisture content, G genotype, N nitrogen. Values followed by the same letter in a column within each N level treatment are not
significantly different between the two genotypes at the 5 % level. ns not significant; * significant at p≤0.05, ** significant at p≤0.01,
***
significant at p≤0.001
N levels and soil moisture treatments (data not shown).
At WD with 25 % w/w of SMC, shoot dry weight was
significantly higher in CSSL50 than in Nipponbare by
13.6 and 10.8 % for the 60 and 120 mg N rootbox−1,
respectively. There was no significant difference in
shoot dry weight between the two genotypes at
30 mg N rootbox−1. The water use of plant per day
was significantly higher in CSSL50 by 13.5, 22.9 and
16.3 % than Nipponbare at 30, 60 and 120 mg N
rootbox −1 , respectively. The total root length of
CSSL50 was significantly greater than those of
Nipponbare by 7.7 %, 15.9 % and 10.0 % at 30, 60
and 120 mg N rootbox−1, respectively. The greater total
root length in CSSL50 was due to the combination
nodal root number, nodal root length and lateral root
length, which were significantly greater than those in
Nipponbare regardless of N application levels
(Supplementary Table S1).
At WD 20 % w/w SMC, CSSL50 had significantly
greater shoot dry weight by 7.3, 12.1 and 10.2 %,
higher water use by 24.6, 46.5 and 34.6 % and
longer total root length by 11.0 %, 13.5 % and
12.9 % than Nipponbare at 30, 60 and 120 mg N
rootbox−1, respectively.
Plant Soil (2015) 386:65–76
Experiment 2 (pot)
Responses in shoot and root growth, and water use
to water deficit as affected by different N forms
application
The shoot dry weight, total and lateral root length,
photosynthetic rate and water use under CWL
(control) and WD (20 % SMC w/w) conditions applied
with different N forms are presented in Table 4. The
ANOVA showed consistent and significant effects of
SMC, N form and G factors on all of those traits. There
were significant interactions between SMC and N form,
and between SMC and G in all of the traits indicating
that the extent of responses varied with treatment combinations. On the other hand, there were no significant
interactions between N form and G and among SMC, N
form and G factor on all of the traits.
Under both soil moisture treatments, the shoot dry
weight, total root length, lateral root length, water use
and photosynthetic in both genotypes tended to be
greater under A and ADCD treatments than under AN,
ANDCD, and N, NDCD treatments (Table 4).
Under CWL, the shoot dry weight, total root length,
lateral root length, photosynthetic rate, water use
(Table 4) and water use efficiency (data not shown) were
similar between Nipponbare and CSSL50 regardless of
N form treatments. On the other hand, under WD at
20 % w/w of SMC, CSSL50 had significantly greater
shoot dry weight by 41.2–56.6 %, total root length by
37.1–45.3 %, lateral root length by 38.0–61.4 %, photosynthetic rate by 15.6–24.8 %, and water use by 25.0–
30.7 % than Nipponbare (Table 4) while water use
efficiency was not significantly different between the
two genotypes (data not shown) regardless of N forms
applied. Furthermore, under WD condition at 20%w/w
of SMC, the difference in total root length between
CSSL50 and Nipponbare did not vary among N forms.
Discussion
This study evaluated the combined effects of water
supply and N levels (Experiment 1) as well as water
supply and N form treatments (Experiment 2) on the
whole root system development and water uptake in rice
to examine if the expression of root system plasticity
triggered by mild drought stress and its contribution to
water uptake would be affected by nitrogen application
71
levels and forms. Both experiments showed that there
was a significant effect of SMC, N (i.e. the levels of N
application in Experiment 1 and the forms of N treatment in Experiment 2) and G on shoot growth, water
use, photosynthetic rate, and root system development
(Tables 3–4 and Fig. 1). Moreover, those traits were
significantly greater in CSSL50 than in Nipponbare
regardless of N levels and forms under water deficit
conditions but not under CWL conditions, thus an observed significant SMC by G interaction. Furthermore,
in Experiment 1, there was a significant interaction
between SMC and N level where the increase in N
levels increased the shoot and root growth and water
use at different extent depending on water supply.
Similarly, a significant interaction between SMC and
N form in Experiment 2 indicated that the magnitude
responses of shoot and root growth and water uptake to
N form treatments differed between WD and CWL
conditions. However, regardless of soil moisture conditions, both CSSL50 and Nipponbare showed greater
shoot and root growth in both the ammonium and mix
of ammonium and nitrate treatments than in nitrate
treatment regardless of the addition of DCD (Table 4).
These results implied that both CSSL50 and
Nipponbare preferred ammonium more than nitrate nutrition, and thus no significant interaction was observed
between N form and G (Table 4) which is similar to
those found by Gao et al. (2010).
Response to water deficit and N level treatments
The root box-pinboard method is indispensable for
collecting the whole root system with minimal damage
which preserved the resulting architecture as regulated
by imposed soil moisture conditions, and thus the contribution of root system development to plant water use
can be precisely evaluated (Kono et al. 1987; Suralta
et al. 2010; Kano-Nakata et al. 2012). In this study, the
whole root system was evaluated based on nodal and
lateral root production (Supplementary Table S1,
Fig. 1). Under WD conditions at 20 and 25 % w/w of
SMC, CSSL50 had significantly greater length of nodal
and lateral roots than Nipponbare across different N
levels (Supplementary Table S1). The differences in
total nodal and lateral root lengths between the two
g en o t y p es , h ow ev e r, v ar i e d w i t h N l e ve l s .
Consequently, CSSL50 had longer total root length than
Nipponbare, and such differences were more evident at
high than low N level (Table 3). These results confirm
72
Plant Soil (2015) 386:65–76
Table 4 Shoot dry weight (SDW, g plant−1), total root length
(TRL, m plant−1), lateral root length (LRL, m plant−1), photosynthetic rate (Pn, μmol m−2 s−1) and water use (WU, g plant−1 day−1)
Soil moisture content
N form
CWL
A
ADCD
N
NDCD
AN
ANDCD
20 % w/w
A
ADCD
N
NDCD
AN
ANDCD
of Nipponbare and CSSL50 grown under continuously waterlogged (CWL) and water deficit (WD) conditions at 20 % w/w
of SMC as affected by N form treatments (Experiment 2)
Genotype
SDW (g plant−1)
TRL (m plant−1)
LRL (m plant−1)
WU (g plant−1 day−1)
Pn (μmol m−2 s−1)
27.8a
Nipponbare
12.6a
93.9a
33.4a
64.1a
CSSL50
12.4a
92.8a
32.9a
62.6a
28.1a
Nipponbare
12.8a
94.3a
33.6a
68.3a
29.1a
CSSL50
12.7a
94.0a
33.7a
65.5a
28.9a
Nipponbare
9.8a
65.7a
21.5a
47.7a
23.9a
CSSL50
10.0a
67.3a
21.8a
50.6a
22.0a
Nipponbare
9.9a
67.6a
23.3a
48.8a
22.7a
CSSL50
9.4a
64.4a
18.9a
47.9a
22.6a
24.6a
Nipponbare
10.8a
74.0a
24.1a
60.3a
CSSL50
10.5a
72.1a
21.1a
55.7a
25.9a
Nipponbare
10.7a
73.a
23.1a
62.4a
25.5a
CSSL50
11.1a
73.5a
24.2a
58.6a
25.6a
Nipponbare
5.0b
45.1b
22.7b
35.1b
21.1b
CSSL50
7.7a
62.3a
33.5a
45.0a
26.4a
Nipponbare
5.6b
46.5b
23.2b
38.7b
22.4b
CSSL50
8.5a
64.8a
35.2a
49.2a
27.1a
Nipponbare
3.9b
39.9b
23.0b
27.8b
18.5b
CSSL50
5.8a
58.0a
35.9a
34.8a
22.3a
119b
Nipponbare
3.8b
40.4b
23.7b
26.4b
CSSL50
5.9a
55.4a
32.7a
33.7a
22.0a
Nipponbare
4.7b
42.6b
21.0b
31.7b
19.7b
CSSL50
6.6a
59.3a
32.5a
41.1a
23.6a
Nipponbare
4.8b
42.8b
20.7b
33.9b
20.2b
7.0a
60.6a
33.5a
42.6a
23.9a
SMC
CSSL50
***
***
*
***
***
N form
***
***
***
***
***
G
***
***
***
***
***
SMC x N form
*
***
***
*
*
SMC x G
***
***
***
***
***
G x N form
ns
ns
ns
ns
ns
SMC x N form x G
ns
ns
ns
ns
ns
A, NH4+ -N alone; ADCD, NH4+ -N with dicyandiamide; N, NO3− -N alone; NDCD, NO3− -N with dicyandiamide; AN, combined NH4+ -N
and NO3− -N; ANDCD, combined NH4+ -N and NO3− -N with dicyandiamide
SMC soil moisture content, G genotype, N nitrogen
Values followed by the same letter in a column within each N form treatment are not significantly different between the two genotypes at the
5 % level
ns not significant; *significant at p≤0.05, **significant at p≤0.01, ***significant at p≤0.001
our previous studies under field condition that N application enhanced the expression of developmental plasticity of root system triggered by mild drought stress
condition and N application interaction (Tran et al.
2014).
The plants have the ability to survive water deficit
condition through drought (dehydration) avoidance
mechanism by maintaining both water (Serraj et al.
2009; Gowda et al. 2011) and nutrient uptake from the
drying soil (Suralta 2010). In this study, we precisely
Plant Soil (2015) 386:65–76
30 mg N roottbox-1
73
60 mg N rootbox-1
120 mg N rootbox-1
Fig. 1 Root system profiles of Nipponbare (A-I), CSSL50 (J-R)
grown under continuously waterlogged (A, J, D, M, G, P) and
water deficit conditions at 25 % w/w of SMC (B, K, E, N, H, Q),
and 20 % w/w of SMC (C, L, F, O, I, R) as affected by the amount
of N applied (30 mg N rootbox−1(A, B, C; J, K, L), 60 mg N
rootbox−1 (D, E, F; M, N, O) and 120 mg N rootbox−1 (G, H, I; P,
Q, R) for 38 DAS. Root systems were sampled with rootboxpinboad method (Kono et al. 1987; Kano-Nakata et al. 2012)
(Experiment 1). Bars =5 cm. CWL, continuously waterlogged;
WD, water deficit
measured the water use and found that the increased root
length of plants grown under WD conditions promoted
water uptake which was evident in CSSL50 but not in
Nipponbare although the difference in water use due to
total root length varied with N level treatments
(Tables 3, 4). High level of N application markedly
increased the differences in water use between
CSSL50 and Nipponbare (Table 3) due to the greater
expression of root plasticity in CSSL50 than in
Nipponbare (Table 4). On the other hand, water use
efficiency was not significantly different regardless of
genotypes and levels of N application (data not shown).
This indicates that dry matter production is a function of
water use but not water use efficiency and therefore the
adaptation of rice under water-limited conditions in
terms of growth and productivity could be achieved
mainly through dehydration avoidance by maintaining
water uptake rather than by tolerance to desiccation
(Blum 2005), which was consistently attributed to root
plasticity expression under progressive drought stress
(Kato et al. 2007; Siopongco et al. 2008; Suralta et al.
2010, Kano-Nakata et al. 2011).
Response to water deficit and N form treatments
It was previously shown that N forms affected root dry
weight (Yang et al. 2012), root volume (Guo et al. 2007;
Gao et al. 2010), total root length and root surface area
(Gao et al. 2010) in rice but the extent of effects depends
on cultivars (Song et al. 2011). Under aerobic condition,
the prevalent form of available N is nitrate and/or the
mixture of ammonium and nitrate (Guo et al. 2007). The
results of Experiment 2 showed that under 20 % w/w of
SMC, CSSL50 showed a consistently and significantly
74
Plant Soil (2015) 386:65–76
greater shoot dry matter production than Nipponbare
regardless of N forms, which was partly attributed to
their genotypic differences in tillering (data not shown).
The differences in shoot dry matter production were also
attributed to the greater ability of CSSL50 than
Nipponbare to maintain photosynthesis (Table 4) and
stomatal conductance (data not shown) as a result of
greater water uptake supported by greater root system
development (Table 4). The above results were consistent with those found in Experiment 1. Moreover, root
system developmental responses of the two genotypes
were similar among different N forms (Fig. 2). The
results imply that the expression of plasticity in root
system development in CSSL50 was triggered by water
deficit conditions but not by the parallel changes in N
form supply. This CSSL is currently being used in
pinpointing the genetic control of root plasticity under
mild drought stress. Based on the above result, it is
highly probable that the potential QTLs associated with
root plasticity are expressed in response to mild moisture deficit conditions rather than to the changes in N
form supply.
When NH4+-N is added to the soil, it will undergo
nitrification process, whereby soil bacteria called
nitrosomonas convert the ammonium to nitrite and then
to nitrate. The use of a nitrification inhibitor such as
DCD retards the conversion of ammonium to nitrate
(Bolan et al. 2004). In this study, the amounts of
NH4+-N in soil were higher at ADCD and ANDCD
treatments than A and AN treatments under both CWL
and WD conditions (Table 1 and 2) which indicate that
Total root length (m plnat-1)
80
17.2 m
18.3 m
18.1 m
DCD inhibited the conversion of ammonium to nitrate
but did not affect the plant growth (data not shown), as
also found by Li et al. (2009). Under CWL, NH4+-N
was the only N form available in soil when applied with
N (urea) fertilizer (Table 1) or ammonium fertilizer
(Table 2), while NO3−-N was not detected under any
of N level (Table 1, Experiment 1) nor N form application (Tables 2, Experiment 2). In contrast, under WD
conditions, the amount of NO3−-N in the soil increased
when N fertilizer application was increased (Table 1).
Also, the amount of NO3−-N in soil was higher than that
of NH 4 + -N when N was applied in nitrate form
(Table 2), similar to the findings of Aulakh et al.
(2000). However, the amount of NO3−-N and NH4+-N
available in the soil (Table 2) did not influence the
CSSL50’s greater root system development than
Nipponbare (data not shown). These results imply that
the expression of plasticity in root system development
triggered by mild drought stress condition was independent of N forms and the amounts of NO3−-N that is the
prevalent form of N under aerobic soil conditions.
Conclusion
The results indicate that the expression of root plasticity
as shown by the differences in root system development
between CSSL50 and Nipponbare increased the water
uptake under mild WD conditions with higher level of N
application. The resulting root plasticity was due to the
greater ability of CSSL50 to maintain root production
15.0 m
16.7 m
17.8 m
60
40
20
0
A
N
AN
ADCD
N form treeatments
Fig. 2 Total root length of CSSL50 (☐) and Nipponbare (■) under
water deficit conditions at 20 % w/w of SMC as affected by N
form treatments (Experiment 2). Numbers above the bars indicate
the differences in total root length between the two genotypes
NDCD
A
ANDCD
under each N form treatments. A, NH4+-N alone; ADCD, NH4+N with dicyandiamide; N, NO3−-N alone; NDCD, NO3−-N with
dicyandiamide; AN, combined NH4+-N and NO3−-N; ANDCD,
combined NH4+-N and NO3−-N with dicyandiamide
Plant Soil (2015) 386:65–76
and elongation than Nipponbare via the maintenance in
nodal root production and promoted lateral root production, which increased soil water extractions. The increased soil water extractions then resulted in the maintenance of photosynthesis and ultimately dry matter
production. On the other hand, the root plasticity expression under mild WD was only enhanced by level of
N application but not by forms of N. These indicate that
the expression of root plasticity was generally in response to moisture deficit but not due to the effect of
the changes in N form available. The root plasticity
expressions observed in the field, was precisely quantified under greenhouse condition including its contributions to water uptake, which were made possible by the
use of rootbox and pot experiments.
Acknowledgments We thank Dr. Jonathan M. Niones of the
Philippine Rice Research Institute for a critical review and useful
comments on our manuscript. This research was funded by the
Grant-in-Aid for Scientific Research (No.22405042) from the
Japan Society for the Promotion of Science, and partially supported by the Japan Science and Technology Agency (JST)/Japan
International Cooperation Agency (JICA), the Science and Technology Research Partnership for Sustainable Development
(SATREPS).
References
Allahyar F (2011) Interaction effects of nitrogen and irrigation
methods on the growth and yield of rice in Amol area. Intl J
Agri Crop Sci 3–4:111–113
Aulakh MS, Khera TS, Doran JW (2000) Mineralization and
denitrification in upland, nearly saturated and flooded subtropical soil. I. Effect of nitrate and ammoniacal nitrogen.
Biol Fertil Soils 31:162–167
Bañoc DM, Yamauchi A, Kamoshita A, Wade LJ, Pardales JR Jr
(2000) Genotypic variations in response of lateral root development to fluctuating soil moisture in rice. Plant Prod Sci 3:
335–343
Belder P, Bouman BAM, Spiertz JHJ, Peng S, Castañeda AR,
Visperas RM (2005) Crop performance, nitrogen and water
use in flooded and aerobic rice. Plant Soil 273:167–182
Blum A (2005) Drought resistance, water use efficiency and yield
potential are they compatible, dissonant and mutually exclusive? Aust J Agr Res 56:1159–1168
Bolan NS, Saggar S, Singh J (2004) The role of inhibitors in
mitigating nitrogen losses in grazed pasture. N Z Soil News 42
Castillo EG, Tuong TP, Singh U, Inubushi K, Padilla J (2006)
Drought response of dry-seeded rice to water stress timing and
N-fertilizer rates and sources. Soil Sci Plant Nutr 52:496–508
Dorlodot S, Forster B, Pagès L, Price A, Tuberosa R, Draye X
(2007) Root system architecture: opportunities and constraints for genetic improvement of crops. Trends Plant Sci
12:474–81
75
Fageria NK (2010) Root growth of upland rice genotypes as
influenced by nitrogen fertilization. 19th World Congress of
Soil Science, Soil Solutions for a Changing World, Australia,
pp 120–122
Gao Y, Li Y, Yang X, Shen Q, Li H, Guo S (2010) Ammonium
nutrition increases water absorption in rice seedlings (Oryza
sativa L.) under water stress. Plant Soil 331:193–201
Gowda VRP, Henry A, Yamauchi A, Shashidhar HE, Serraj R
(2011) Root biology and genetic improvement for drought
avoidance in rice. Field Crop Res 122:1–13
Guo S, Kaldenhoff R, Uehlein N, Sattelmacher B, Brueck H
(2007) Relationship between water and nitrogen uptake in
nitrate- and ammonium-supplied Phaseolus vulgaris L.
plants. J Plant Nutr Soil Sci 170:73–80
IRRI (2009) CROPSTAT Version 7.2. Metro Manila: International
Rice Research Institute. Available at http://archive.irri.org/
science/software/cropstat.asp
Kamoshita A, Rodriguez R, Yamauchi A, Wade LJ (2004)
Genotypic varia- tion in response of rainfed lowland rice to
prolonged drought and rewatering. Plant Prod Sci 7:406–420
Kamoshita A, Wade LJ, Yamauchi A (2000) Genotypic variation
in response of rainfed lowland rice to drought and rewatering.
III. Water extraction during the drought period. Plant Prod Sci
3:189–196
Kano M, Inukai Y, Kitano H, Yamauchi A (2011) Root plasticity
as the key root trait for adaptation to various intensities of
drought stress in rice. Plant Soil 342:117–128
Kano-Nakata M, Inukai Y, Wade LJ, Siopongco JDLC, Yamauchi
A (2011) Root development and water uptake, and shoot dry
matter production under water deficit conditions in two
CSSLs of rice: functional roles of root plasticity. Plant Prod
Sci 14:307–317
Kano-Nakata M, Suralta RR, Niones JM, Yamauchi A (2012)
Root sampling by using a root box–pinboard method.
In: Shashidhar HE, Henry A, Hardy B (eds)
Methodologies for root drought studies in rice. Los
Baños (Philippines), International Rice Research
Institute, pp 3–8
Kano-Nakata M, Gowda VRP, Henry A, Serraj R, Inukai Y, Fujita
D, Kobayashi N, Suralta RR, Yamauchi A (2013) Functional
roles of the plasticity of root system development in biomass
production and water uptake under rainfed lowland conditions. Field Crop Res 144:288–296
Kato Y, Kamoshita A, Yamagishi J, Imoto H, Abe J (2007) Growth
of rice (Oryza sativa L.) cultivars under upland conditions with different levels of water supply. Plant Prod
Sci 10:3–13
Kondo M, Sing CV, Agbisit R, Murty MVR (2005) Yield response
to urea and controlled-release urea as affected by water
supply in tropical upland rice. J Plant Nutr 28:201–219
Kono Y, Yamauchi A, Nonoyama T, Tatsumi J, Kawamura N
(1987) A revised experimental system of root-soil interaction
for laboratory work. Environ Contor Biol 25:141–151
Landi P, Giuliani S, Salvi S, Ferri M, Tuberosa R, Sanguineti MC
(2010) Characterization of root-yield-1.06, a major constitutive QTL for root and agronomic traits in maize across water
regimes. J Exp Bot 61:3553–3562
Laperche A, Devienne-Barret F, Maury O, Le Gouis J, Ney B
(2006) A simplified conceptual model of carbon/nitrogen
functioning for QTL analysis of winter wheat adaptation to
nitrogen deficiency. Theor Appl Genet 113:1131–1146
76
Li Y, Gao Y, Ding L, Shen Q, Guo S (2009) Ammonium enhances
the tolerance of rice seedlings (Oryza sativa L.) to drought
condition. Agr Water Manag 96:1746–1750
Nevo E, Chen G (2010) Drought and salt tolerances in wild relatives
for wheat and barley improvement. Plant Cell Environ 33:
670–685. doi:10.1111/j.1365-3040.2009.02107.x
Niones JM, Suralta RR, Inukai Y, Yamauchi A (2012) Field
evaluation functional roles of root plastic responses on dry
matter production and grain yield of rice under cycles of
transient soil moisture stresses using chromosome segment
substitution lines. Plant Soil 359:107–120
Niones JM, Suralta RR, Inukai Y, Yamauchi A (2013) Role of root
aerenchyma development and its associated QTL in dry
matter production under transient moisture stress in rice.
Plant Prod Sci 16:205–216
Obara M, Tamura W, Ebitani T, Yano M, Sato T, Yamaya T
(2010) Fine-mapping of qRL6.1, a major QTL for root
length of rice seedlings grown under a wide range of
NH4 + concentrations in hydroponic conditions. Theor
Appl Genet 121:535–547
Otoo E, Ishii R, Kumura A (1989) Interaction of nitrogen supply
and soil water stress on photosynthesis and transpiration in
rice. Jpn J Crop Sci 58:424–429
Price AH, Steele KA, Moore BJ, Barraclough PB, Clark LJ (2000)
A combined RFLP and AFLP linkage map of upland rice
(Oryza sativa L.) used to identify QTLs for root-penetration
ability. Theor Appl Gen 100:49–56
Qian X, Shen Q, Xu G, Wang J, Zhou M (2004) Nitrogen Form
Effects on Yield and Nitrogen Uptake of Rice Crop Grown in
Aerobic Soil. J Plant Nutr 27:1061–1076
Ruta N, Liedgens M, Fracheboud Y, Stamp P, Hund A (2010) QTLs
for the elongation of axile and lateral roots of maize in response
to low water potential. Theor Appl Genet 120:621–631
Serraj R, Krishnamurthy L, Kashiwagi J, Kumar J, Chandra S,
Crouch JH (2004) Variation in root traits of chickpea (Cicer
arietinum L.) grown under terminal drought. Field Crop Res
88:115–127
Serraj R, Kumar A, McNally KL, Slamet-Loedin I, Bruskiewich
R, Mauleon R, Cairns J, Hijmans RJ (2009) Improvement of
drought resistance in rice. Adv Agron 103:41–99
Sharma S, Xu S, Ehdaie B, Hoops A, Close TJ, Liukaszewskie AJ,
Giles Waines J (2011) Dissection of QTL effects for root
traits using a chromosome arm-specific mapping population
in bread wheat. Theor Appl Genet 122:759–769
Siopongco JDLC, Yamauchi A, Salekdeh H, Bennett J, Wade LJ
(2005) Root growth and water extraction responses of double
haploid rice lines to drought and rewatering during the vegetative stage. Plant Prod Sci 9:141–151
Siopongco JDLC, Yamauchi A, Salekdeh H, Bennett J, Wade LJ
(2006) Growth and water use response of doubled haploid
Plant Soil (2015) 386:65–76
rice lines to drought and rewatering during the vegetative
stage. Plant Prod Sci 9:141–151
Siopongco JDLC, Sekiya K, Yamauchi A, Egdane J, Ismail AM,
Wade LJ (2008) Stomatal responses in rainfed lowland rice to
partial soil drying; evidence of root signals. Plant Prod Sci 11:
28–41
Song W, Makeen K, Wang D, Zhang C, Xu Y, Zhao H, Tu E,
Zhang Y, Shen Q, Xu G (2011) Nitrate supply affects root
growth differentially in two rice cultivars differing in nitrogen
use efficiency. Plant Soil 343:357–368
Suralta RR (2010) Plastic root system development responses to
drought-enhanced nitrogen uptake during progressive soil
drying conditions in rice. Philipp Agr Sci 93:458–462
Suralta RR, Inukai Y, Yamauchi A (2010) Dry matter production
in relation to root plastic development. Oxygen transport, and
water upatake of rice under transient soil moisture stresses.
Plant Soil 332:87–104
Tran TT, Kano-Nakata M, Takade M, Menge D, Mitsuya S, Inukai
Y, Yamauchi A (2014) Nitrogen application enhanced the
expression of developmental plasticity of root system triggered by mild drought stress in rice. Plant Soil. doi:10.1007/
s11104-013-2013-5
Uga Y, Okuno K, Yano M (2011) Dro1, a major QTL involved in
deep rooting of rice under upland field conditions. J Exp Bot
62:2485–2494
Wade LJ, Fukai S, Samson BK, Ali A, Mazid MA (1999) Rainfed
lowland rice: physical environment and cultivar requirements. Field Crop Res 64:3–12
Wang H, Yamauchi A (2006) Growth and function of roots under
abiotic stress soils. In: Huang B (ed) Plant environment
interactions, 3rd edn. CRC Press, Taylor and Francis
Group, LLC, New York, pp 271–320
Weatherburn M (1967) Phenol-hypochlorite reaction for determination of ammonia. Anal Chem 39:971–974
Yadav RS, Sehgal D, Vadez V (2011) Using genetic mapping and
genomics approaches in understanding and improving
drought tolerance in pearl millet. J Exp Bot 62:397–408
Yamauchi A, Kono Y, Tatsumi J (1988) Comparative growth
analysis of upland rice and maize grown under different soil
moisture conditions. Jpn J Crop Sci 57:174–183
Yamauchi A, Pardales JR Jr, Kono Y (1996) Root system structure
and its relation to stress tolerance. In: Ito O, Katayama
K, Johansen C, Kumar Rao JVDK, Adu-Gyamfi JJ,
Rego TJ (eds) Roots and nitrogen in cropping systems
of the semi-arid tropics. JIRCAS Publication, Tsukuba,
Japan, pp 211–234
Yang X, Li Y, Ren B, Ding L, Gao C, Shen Q, Guo S (2012)
Drought-induced root aerenchyma formation restricts water
uptake in rice seedlings supplied with nitrate. Plant Cell
Physiol 53:495–504