Journal of Ravishankar University –B,
32 (1), 43-60 (2019)
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Identification
of QTLS for NH4+ and NO3- use efficiency under
water stress and non-stress conditions and expression analysis of glutamine
synthetase and nitrate reductase in rice (Oryza Sativa L.)
Rashmi Upadhyay*
Indira Gandhi Krishi Vishwavidyalaya, Raipur, C.G, India.
*Corresponding
Author: rashmiupadhyay123@gmail.com
[Received: 31 January 2019; Revised
version: 27 April 2019; Accepted: 28 April 2019]
Abstract. Nitrogen
(N) is one of the most critical inputs and the current average nitrogen use
efficiency (NUE) in the rice field is approximately 33%, poorest among cereals.
Predominant form of N in aerobic soils is nitrate (NO3-)
while ammonium (NH4+) exists in anaerobic soils.
Development of cultivars with improved NH4+ or NO3-
use efficiency by harnessing inherent significant variability for NUE can be an
important approach. Considering these facts, the present study was established
with one hundred twenty two and selected thirty two recombinant inbred lines
(RILs) of two indica genotypes, Danteshwari × Dagad deshi under three nitrogen
forms and three environments. The trend analysis of NH4+-N
& NO3--N dynamics revealed that NH4+-N
concentration persisted more under anaerobic condition and NO3--N
concentration under aerobic conditions. Three way-ANOVA showed high level of
significance for variance components (G, N, E) and their interactions effects
(GXN, GXE, NXE, EXNXG) for yield & NUE and their component traits. Mean
performance of genotypes depicted higher values for agronomically important
traits i.e. yield and NUE under NH4+ as compared
to NO3--N and N0. The phenotypic and genotypic
data was statistically analyzed for QTLs identification for yield & NUE
traits. A total of 58 QTLs conferring the corresponding five traits were
detected under three N forms and two environments. We also investigated the
different members of AMT (Ammonium transporters), NRT (Nitrate transporters),
GS (Glutamine Synthetase) & GOGAT (Glutamate Synthase) genes, involved in
NUE and analyzed the expression pattern of each gene using gene-specific primer
in young rice seedlings. Collectively, OsGln1;1, OsGln1;2, OsGln1;3,
OsGln2, OsGlt1 and OsGlt2 manifested different and
reciprocal responses to nitrate and ammonium supply. The activity of enzymes
NR, NiR, GS & GOGAT was significantly affected by NH4+and
NO3- treatment. These results assist us to identify NH4+
& NO3- responsive cultivars which could be used
for cultivation and/or used as parent’s in future breeding program to produce
better nitrogen use efficiency varieties under water stress and non-stress
conditions.
Key words: ammonium transporters; nitrate transporters; NUE;
glutamine synthetase.
Introduction
The main driver for crop improvement over the years has
been yield. Over the period, the rate of yield improvement has accelerated
primarily not only due to the introduction of an increasingly scientific
approach, but also through external use of fertilizers. Among these fertilizers
in spite of being the most abundant element in the atmosphere (78 %), N is one
of the most limiting nutrients in natural and agricultural ecosystems. The
majority of plant-useable N is consumed as nitrate (NO3-) from well-aerated soils and as
ammonium (NH4+) from poorly aerated, submerged soils. Since rice is capable
of assimilating both forms of N it is adapted to aerobic as well as anaerobic
growth conditions (Kronzucker et al. 1998).
Furthermore, scarcity of water is a
severe environmental constraint to plant productivity. Drought-induced loss in
crop yield probably exceeds losses from all other causes, since both the
severity and duration of the stress are critical. Influence of
drought on plant nutrition may also be related to limited availability of
energy for assimilation of NO3-/NH4+,
PO43-and SO24 - they must be
converted in energy-dependent processes before these ions can be used for
growth and development of plants (Grossman and Takahashi, 2001).
Nitrogen use efficiency (NUE) of a
plant defines its ability to utilize available nitrogen (N) resources to optimize
its productivity. Nitrogen uptake, assimilation and redistribution within cell
along with balance between storage and current use at cellular and whole plant
level are included in this (Abdin et al., 2005). NUE is a matter of
great concern. Burgeoning population of world needs crop genotypes showing
direct relationship with yield and are highly nitrogen responsive. NUE is a
complex trait which is governed by activities of ammonium and nitrate
transporters that are regulated by N form and concentration affects the N
acquisition by roots (Garnett et al., 2009). Intrinsic aspects of N
utilization includes many gene families, including NO3-
and NH4+ transporters genes and primary assimilatory genes that have been
identified by different approaches. To cope with varying NO3-
& NH4+ concentrations in soils, N uptake in roots is mainly
regulated by a high affinity transport system (HATs) that regulates uptake at N
levels <1mM, and a low affinity transport system (LATs) that functions at
high N concentrations >1mM (Glass et al., 2001). Nitrate transporter
system along with nitrate reductase (NR) and nitrite reductase (NiR) enzymes
subsequently facilitate reduction of NO3- in to NH4+. Expression of the NRTs is highly
regulated (Feng et al., 2011). NH4+ from both the nitrate reduction
pathway and direct absorption are subsequently incorporated into amino acids
through the synthesis of glutamine and glutamate (Campbell, 2002) primarily in
the chloroplasts and plastids via the glutamine synthetase (GS)/glutamate synthase
(GOGAT) cycle (Andrews et al., 2004).
Nitrogen
utilization within rice plants followed by NH4+
uptake and assimilation in the roots is a complex process that depends on many
factors during the growth and development of plants. Reverse genetics is a powerful
approach to obtain conclusive evidence on the function of the corresponding
gene products, also enzymes involved in metabolic of nitrogen assimilation
pathway can be characterized by reverse genetics. But now it is very difficult
to identify target genes that are involved in regulation, unlike enzymes in
metabolic pathway, so an approach of quantitative trait loci (QTLs) analysis
could be one way to isolate regulatory genes. Many gene families, including NO3- and NH4+
transporters and primary assimilation genes, amino acid transporters, as well
as transcription factors and other regulatory genes, have been identified by
different approaches. With the identification of orthologous genes from rice,
opportunities are now emerging for utilizing these genes in marker-assisted
breeding for N efficiency (Kant et al. 2011). So in rice, the effect of
QTLs would be potential research to confirmed target genes controlling uptake,
assimilation, and metabolism of nitrogen, as well as nitrogen use efficiency. The highly
complex objective requires comprehensive knowledge and deep understanding of
the physiological, biochemical and molecular responses of rice genotypes under
different nitrogen regimes. In the present investigation, attempts has been
made to identify efficient lines in mapping population derived from Indica
parents that shows differential expression of transporter systems and key
assimilatory enzymes along with biochemical characterization of these enzymes
& morphological characterization of their root system.
Experimental
Plant material
and experimental site
The plant material used in present
study includes two parent viz., Danteshwari and Dagad deshi and their
recombinant inbred lines (RILs) population. Phenotypic data was generated on
Vertisol of Research cum Instructional farm, College of Agriculture, Indira
Gandhi Krishi Vishwavidyalaya, Raipur. Molecular Marker Laboratory, Department
of Plant Molecular Biology and Biotechnology, IGKV, Raipur was used as platform
to generate the genotypic data for identifying QTLs for agronomically complex
trait i.e. NUE, study NH4+ and NO3- dynamics in soil and
carry out enzyme assay and expression analysis of key known genes.
Methods
Field Studies
The RIL lines derived from a cross
between Danteshwari×Dagad deshi were evaluated in the field during wet season
2014, 2015 and summer 2015-16 at research cum instructional farm of College of
Agriculture, IGKV, Raipur. The rainfed and TSD fields selected for the study
were upland in topology with good drainage and percolation rate. In case of
terminal drought trials to reduce the chance of rainfall interfering with
drought development in the wet season, trials were planted later and water was
drained from the field so that the crop has a better chance of being exposed to
drought. Good bunds were a prerequisite for this experiment to limit seepage
and treatment flow. The entire experiment was laid out in factorial randomized
block design with two replication of different forms of nitrogen under varied
sets of conditions. The field view of experiment is presented in Fig.1.
Evaluating NH4+-N and NO3- -N
dynamics in soil
On-farm experiment conducted during wet
season of 2014, 2015 was subjected to analysis of soil nitrogen inorganic
fraction in each treatment under all environments.
Nitrogen
inorganic fractions
The fractionation of soil nitrogen was
carried out by the procedure given by Bremner and Keeney (1965), described by
Keeney and Nelson (1982). Steps involved in the process are elaborated as such:
Extraction of
NO3--N and NH4--N with 2.0 M KCl
Soil moisture determination
Clean and dry tin + lid were weigh to
0.01 g (W1). 30g of representative moist soil was selected and sample was
placed in the weighing tin and lid was replaced. Tin and contents was weigh to
0.01g (W2). Lid was removed and tin was placed with contents in the oven and
dry to constant weight between 105 °C and 110 °C Tin with contents was removed
from oven and placed as whole in the desiccator to cool. Tin and
content was weighed finally.
The soil moisture was calculated in
percent by formula:
Where: SMC = Soil moisture content (%), W1 = Weight of
tin (g), W2 = Weight of moist soil + tin (g), W3 = Weight of dried soil + tin
(g)
Preparation of equilibrium extract
Principle
Ammonium in held in exchangeable form
in soils in the same manner as exchangeable metal cations. Fixed or non
exchangeable NH4+ can make up a significant portion of soil N; however
fixed NH4+ is defined as the NH4+ in soil that cannot be replaced by the
neutral K salt solution. Exchangeable NH4+ is extracted by shaking with 2.0 M KCl.
Nitrite is water soluble and hence can also be extracted by the 2.0 M KCl.
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Figure 1. A
view of experimental field
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Steps involved
30 g of soil sample was taken in a
polyethylene bottle. 150 ml of 2.0 M KCl solution was added (If the sample is
limited, it can be reduced to a minimum of 1.0 g and 5 ml 2.0 M KCl to keep 1 :
5 ratio which is mandatory). Bottle was
capped and kept in mechanical shaker for 30 min. Soil-KCl suspension was allowed to
settle until the supernatant liquid is clear (usually about 30 minutes). This
supernatant is filtered into Erlenmeyer flasks gravimetrically through filter
paper fixed in funnels. Analyses of NO3--N
and NH4+-N elements was performed on aliquots of this liquid (If the KCl extract
cannot be analyzed within 24 hours after its preparation store the filtrate in
a refrigerator until analyses can be performed).
Determination of NH4+-N
and NO3--N in 2.0 M KCL extract using KEL plus
distillation apparatus
Material
Distillation
apparatus consisting of 500 or 800 mL Kjeldahl flasks, Connecting
bulbs, Vertical
condenser, Hot plates, 300 mL
receiving beakers or Erlenmeyer flasks, Microburette.
Procedure
10 ml of boric acid-indicator solution
was added to 125 ml Erlenmeyer flask marked to indicate a volume of 75 ml and
flask was placed under the condenser of the steam-distillation apparatus so
that the end of the condenser is in the boric acid. An aliquot (usually
20-50 ml) of the soil extract was transferred into a distillation flask and add
0.4 g of MgO was added. Commence distillation by placing plug in the steam
bypass tube of the distillation apparatus and 75 ml of distillate was
collected. End of the condenser was rinsed and ammonium-N in the distillate was
determined by titration with .005N sulphamic acid using a microburette graduated
at 0.02-ml intervals. The colour change at the endpoint is from green or blue
to a permanent pink. Blank titration value is obtained before running
representative samples.After removal of NH4+-N from the sample as described above.
The sample in the distillation flask was treated with 1 ml of (1%) Sulphamic
acid solution and the flask was swirled for a few seconds to destroy nitrite
(NO2-). 0.2-0.4 g of Devardas alloy was added to the
distillation flask and the distillation was continued. The amount of NO3-
-N was determined same as described above for exchangeable ammonical nitrogen
omitting the 2 step. Brief view is given in Fig 2.2.
Analyses of soil testing results
Calculation
NH4+-N / NO3---N
(ppm) = (V-B’) X N X R X 14.01 X 1000
W
- Θ
Where, V= Volume of sulphamic acid required
for titrating sample (ml) B’= Blank titration value (ml), N = Normality of
sulphamic acid, R = ratio between volume of extract obtained and volume used
for titration, W = Weight of oven-dried sample, Θ = Weight of water (g) per 30g
oven-dried sample.
Extraction and assay of nitrogen uptake
and ammonium assimilation enzymes
The three enzymes, namely, NR, NiR, GS and GOGAT were
assayed in freshly harvested leaf at seedling stage of rice genotypes. The
protein was determined from all of the enzyme extracts. All the assays were
done with three replications. Specific activity of an enzyme has been defined
as µmol of product formed per mg protein.
Nitrate Reductase (NR)
The nitrate reductase (NR) activity was
estimated by using the method described by (Hageman and Hucklesby, 1971). 500mg
of freshly harvested leaf tissue were cut into small pieces and were
transferred into test tubes containing 3mL of each 0.2M potassium phosphate buffer (pH 7.5), and 0.4M potassium nitrate
which were then incubated in dark at 33◦C for 30 min. Add 0.2mL of above
extract after incubation in separate test tube containing 1mL distilled water.
Add 1.2mL (1:1 v/v) mixture of NED (0.1% w/v) and sulphanilamide (1% (w/v) in
3N HCl) and keep in darkness for 15min for pink colour development. The
absorbance was measured at 540 nm with the help of spectrophotometer using
distilled water as blank, and the amount of nitrite present was found out by
comparing with the standard curve.
Glutamine Synthetase (GS)
The assays were carried out by
continuous spectrophotometric rated determination method. The extraction buffer included, 10mM-Tris HCl (pH 7.6),
1mM-MgCl2, 1mMEDTA, and 1mM-2 mercaptoethanol. Leaves (2g) were grinded using
liquid N2 in the presence of cover slips followed by centrifugation at 12,000xg
for 30min at 4◦C. Supernatant was collected and stored at−20◦C (Pateman, 1969).
Glutamate Synthase (GOGAT)
Standard assay mixture contained 40mM
potassium phosphate buffer (pH 7.5),
10mM L-glutamine, 10mM 2oxoglutarate, 0.14mM NADH, and crude enzyme (final
volume 3m 1). Increase in absorbance at 340nm for 34min at room temperature
(25◦C) was recorded. Absorbance (340nm/min) was calculated from initial linear
portion of the curve (Grot and Vance, 1981).
Stover and grain nitrogen analysis
Stover and
grain nitrogen concentrations were estimated by digestion methods using
MicroKjeldahl’s apparatus (Humphries, 1956) along with H2SO4
containing digestion mixture (10 parts potassium sulphate and 1 part copper
sulphate), 1 g working sample from each treatment from submitted ground sample
were put in a Kjeldhal’s flask and digested. The blank was run and determined
periodically, using all the ingredients except the sample. The percentage of
protein in the grain was calculated by multiplying the grain N content with a
constant factor of 6.25 and results were expressed on dry weight basis.
Temporal
expression analysis of transporter system and assimilatory enzymes by
Quantitative Real Time -PCR analysis (qPCR) In the present investigation qPCR
was performed with selected gene specific primers with actin and tubulin as
internal controls or housekeeping genes. qPCR was accomplished on Mx3000P® QPCR
System (Stratagene, USA) using gene specific primers. qPCR was performed using
SYBR green qPCR mix (Applied biosystems and Thermo Fisher make) using
approximately 700-1000 ng total cDNA in a 20 ul reaction mixture containing
final composition of 1X qPCR mix and 0.5-0.8 uM of each forward and reverse
primers. Reaction was set as per manufacturer’s instructions. Blank was always
set for each primer during every PCR setup. The relative quantification of
expression was done by using the mathematical model given by Livak and
Schmittgen (2001).
R = 2 -∆∆Ct
∆∆Ct= [ΔCt treatment – ΔCt
control]
ΔCt treatment= Ct with
primer-Ct with endogenous control (treatment)
ΔCt control= Ct with
primer-Ct with endogenous control (Control)
QTL analysis
The linkage map was constructed with QTL Cartographer
2.5. Graphical genotyping of these molecular data was done using GGT 2.0 (Van
Berloo, 1999). The phenotypic and genotypic data was analyzed using QTL
cartographer 2.5 (Composite Interval Mapping) with a threshold value of 3.0 LOD
(Wang et al., 2005) and QTL IciMapping 3.2 where a LOD score of 2.5 was
used for declaring the presence of a suggestive QTL (Li et al., 2007).
Statistical
methods
A complete factorial arrangement of
treatments was used (genotype x treatment) as a complete randomized design with
three replications All data was analyzed by analysis of variance, and F-test
was used to determine treatment significance. Duncan’s multiple range test
(DMRT) was used to compare treatment means at 5% probability level using
M-STAT-C 14.2 software. Mean ± standard error mean (SEM) values were calculated
for statistical analysis. Appropriate regression equations and correlation
analysis were also used for further analysis of relations between different
parameters.
Observation and results
Growth parameters
Number of
effective tillers
5 plants from the row of 10 plants were
randomly selected and number of tillers per plants bearing panicle was counted.
Days to 50%
flowering
The time taken (days) from sowing to
flowering period was recorded on plot basis by visual means.
Plant height
Plant height was measured in centimetre
(cm) from soil surface to tip of the tallest panicle at maturity.
Panicle length
Single panicle length of 5 randomly
selected plants was measured in centimetres from base to tip of panicle and
averaged it.
Biological yield
The biological yield was recorded as
actual weight in gram of total biomass of shoot portion per line.
Grain yield
The actual
yield of grain in gram per line was recorded.
Harvest index
Harvest index was worked out by using the formula as given below:
Chlorophyll content
SPAD-502 was
used to measure chlorophyll content of leaves in SPAD units. Readings were
taken of young fully expanded leaves of five representative plants from each
genotype and average reading was recorded.
Results and Discussion
Seasonal NH4+-N and NO3--N
dynamics in soil (kg ha-1)
The trend
analysis of NH4+-N and dynamics during kharif 2014
and kharif 2015 revealed that NH4+-N concentration
persisted more under anaerobic condition as compared to aerobic condition as
compared to aerobic condition while trend analysis of NO3--N
dynamics during kharif 2014 and kharif 2015 showed that maximum
values of NO3--N concentrations under aerobic conditions
as compared to anaerobic conditions under all treatments and soil environments.
The trend analysis can be seen in fig. 3.1 and 3.2.
Yield and NUE related traits
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Figure 2. Periodic
changes in ammonical nitrogen status under different levels of nitrogenous
fertilizer and soil moisture regime during kharif 2014 and 2015
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Yield and yield
related traits were evaluated in 122 and 32 RILs during wet season 2014 and
2015. Three way-ANOVA showed high level of significance for variance components
(G, N, E) and their interactions effects (GXN, GXE, NXE, EXNXG). Nitrogen and
environment was the main consideration of present study and they significantly
affected investigated traits which allow us to conclude that it is possible to
manipulate these plant parameters in favour of higher grain yield by using
appropriate nitrogen form and environment. During wet season 2014, mean
performance of genotypes depicted higher values for agronomically important
traits i.e. grain yield, biological yield, plant height and total
tillers under NH4+ treatment (317.1 g/m2, 998.6 g/m2,
117.1 cm & 361.6 g/m2) followed by NO3-
(311.3 g/m2, 827.7 g/m2, 117.1 cm & 303.6 g/m2)
and N0 (352 g/m2 , 850 g/m2 , 170 cm, 108 g/m2
) treatment under irrigated condition . Furthermore, under rainfed condition
high mean phenotypic values were observed under grain yield, biological yield,
plant height and total tillers in NH4+ treatment (241.1 g/m2, 933
g/m2, 86 cm, 450 g/m2) followed by NO3--treatment
(179 g/m2, 651 g/m2, 86 cm, 401 g/m2) and N0
treatment (57 g/m2, 274 g/m2, 71 cm, 272 g/m2).
Moreover, during wet season 2015, NH4+ form showed highest values for all
evaluated traits then NO3- and N0 forms. High
phenotypic coefficient of variability and genotypic coefficient of variability
was obtained for grain yield (>25.6%), biological yield (>19.9%), harvest
index (>20.1%), seedling biomass (>17.9%) and spikelet sterility
(>27.3%) under all sets of conditions during wet season 2014 and 2015 and
broad sense heritability estimates for the estimated 19 traits during wet
season 2014 ranged from 8.2% to 84.1 % and during wet season 2015 ranged from
24% to 98.4 % under differential N and water regimes. NUE and NUE component
traits ANOVA revealed that genotypic effects and genotype x nitrogen
interaction effects were significantly different for investigated N use
efficiency and its component traits (p<0.05, p<0.01).
During wet season 2015, mean
performance of genotypes depicted higher values for agronomically complex traits i.e. straw nitrogen
content (SNC), grain protein content (GPC), grain nitrogen content (GNC), straw
nitrogen yield (SNY), grain nitrogen yield (GNY), biological nitrogen yield
(BNY), nitrogen harvest index (NHI), N uptake (NUE), N-utilization (NUtE) and
N-use efficiency (NUE) was highest in NH4+ treatment (1.23%, 7.3%, 0.5%, 3.5 g/m2,
2.7 g/m2, 6.3 g/m2, 57%, 0.20 gg-1, 45.2 gg-1,
9.8 gg-1) followed by NO3- treatment (1.1%,
6.8%, 0.47%, 2.6 g/m2, 2.1 g/m2, 4.7 g/m2,
56%, 0.18 gg-1, 49.4 gg-1, 9.3 gg-1) and N0 treatment (1.1%,
6.6%, 0.4%, 2.4 g/m2, 1.7 g/m2, 4.2 g/m2,
56.7%, 0.15 gg-1, 51.6 gg-1, 8.8 gg-1).
Similarly, under rainfed condition, maximum values of mean performance of
genotypes was obtained in NH4+ treatment (1.5%, 8.9%, 0.9%, 2.0 g/m2, 4.1
g/m2, 6.1 g/m2, 5%, 0.22 gg-1, 21.8 gg-1, 5.0 gg-1)
followed by NO3- treatment (1.3%, 8.2%, 0.8%, 1.7 g/m2,
3.1 g/m2, 4.7 g/m2, 34.7%, 0.17 gg-1, 25.1 gg-1,
4.7 gg-1) and N0 treatment (1.2%, 6.9%, 6%, 1.1 g/m2, 1.7 g/m2,
2.8 g/m2, 32.5%, 0.10 gg-1, 30.5 gg-1, 3.3 gg-1).
Results is shown in table 1, 2
and 3.
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Figure 3. Periodic
changes in nitrate nitrogen (NO3--N) status
under different levels of nitrogenous fertilizer and soil moisture regime
during kharif 2014 & 2015
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Enzymatic activities and its relationship with Nitrogen
use efficiency
Table 1. Effect of NH4+, NO3- & N0 treatments
on activities of NR, NiR, GS and GOGAT
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Treatment
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NR b
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NiRc
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GSd
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GOGATe
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Leaves
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NH4+
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0.053 ± 0.004
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0.023 ± 0.001
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0.099 ± 0.006
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0.196 ± 0.014
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NO3-
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1.49 ± 0.197
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0.031± 0.002
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0.123 ± 0.007
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0.280± 0.032
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N0
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0.019 ± 0.001
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0.016 ± 0.001
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0.155 ± 0.012
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0.290 ± 0.050
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Roots
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NH4+
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0.002 ± 0.001
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0.001 ± 0.000
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0.023 ± 0.001
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0.051± 0.003
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NO3-
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0.006 ± 0.001
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0.017 ± 0.001
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0.034 ± 0.001
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0.056± 0.003
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N0
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0.001 ± 0.008
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0.001 ± 0.000
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0.038 ± 0.003
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0.68 0.002
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NR activity expressed in µmol NO2−
formed mg−1 proteins min−1. NiR activity
expressed in µmol NO2− reduced mg−1 proteins
min−1. GS activity expressed in µmol Pi formed mg−1
proteins min−1.bGOGAT activity expressed in
µmol NADH oxidized mg−1 proteins min−1
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In a set of 122 RILs, 32 selected
genotypes were evaluated for NUE & related traits and cluster analysis was
further performed to classify these genotypes as high NUE, medium NUE and low
NUE. Frequency distribution of leaf colour was also recorded to categorize RILs
into four different classes’ viz., dark green, green, light green,
yellow. Investigation of leaf colour trait accompanied by evaluation of NUE,
yield and yield related parameters provided us with the 10 distinct genotypes.
The differences observed in the leaf colour and NUE parameters under different
N forms in these genotypes possibly may be due to genotypic variation in the
activity of enzymes involved in assimilation of nitrogen. Our experiment aimed
to investigate the activity of key assimilatory enzymes namely, Glutamine
Synthetase (GS), Glutamate Synthase (GOGAT), Nitrate Reductase (NR) and Nitrite
Reductase (NiR) at seedling stage in 10 selected rice genotypes. The results
showed that N forms greatly influenced the measured traits and substantial
differences among rice genotypes were recorded in the activities of the enzymes.
Results are shown in Table2and3.
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Table 2. Mean phenotypic performance, range, standard deviation (SD),
Coefficient of variance (CV %) of investigated yield and yield related traits
of 122 RILs and their parents under differential N regimes and environments
during wet season 2014
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Wet Season
2014
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Traits
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N
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IRRIGATED (I)
RILs
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RAINFEED (Ds)
RILs
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D
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DD
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Mean±SEm
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Range
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CV%
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D
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DD
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Mean±SEm
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Range
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CV%
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GY
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NH4+
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625.52
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499.0
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352.1± 14
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37.3-844
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48
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100
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256
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241.1±7
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47- 486
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37
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NO3-
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375
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323
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338.3±10
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55-842
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36
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96
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212
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179.1±8
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15-447
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55
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N0
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320
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242
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312.1±11
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30-673
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37
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41
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79
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57.2±2
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14-165
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48
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BY
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NH4+
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1266
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923
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1065.6±20
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415-1644
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24
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543
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826
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933.1±16
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447-1755
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21
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NO3-
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796
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695
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908.5±20
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392-2561
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28
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500
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810
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651.6±15
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185-1231
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29
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N0
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752
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560
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905.6±13
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492-1496
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17
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225
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324
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274.41±7
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118-515
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30
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HI
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NH4+
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49
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46
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37.4±1
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6.4-56.1
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34
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20
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30
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27.1±0.6
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7.3-56.3
|
30
|
|
|
NO3-
|
47
|
46
|
34.7±0.7
|
11.4-59.6
|
21
|
19
|
26
|
26.0±0.7
|
3.1-42.6
|
32
|
|
|
N0
|
42
|
43
|
32.6±0.8
|
4.9-54.3
|
26
|
17
|
24
|
20.1±0.4
|
8.6-36.1
|
27
|
|
TT/m2
|
NH4+
|
392
|
242
|
361.6±4
|
238-568
|
16
|
483
|
281
|
450.8±6
|
262-636
|
17
|
|
|
NO3-
|
358
|
206
|
303.6±5
|
123-495
|
20
|
393
|
393
|
401±7
|
168-984
|
23
|
|
|
N0
|
190
|
105
|
170.2±2
|
104-271
|
16
|
197
|
258
|
272±4
|
124-464
|
20
|
|
TT/P
|
NH4+
|
8.5
|
5.5
|
7±0.1
|
13.1-5.0
|
20
|
|
|
|
|
|
|
|
NO3-
|
7.8
|
4.3
|
6±0.1
|
11.2-4.0
|
21
|
|
|
|
|
|
|
|
N0
|
5.9
|
3.5
|
5.7±0.1
|
8.8-2.2
|
2
|
|
|
|
|
|
|
ET/P
|
NH4+
|
8
|
5
|
6.4±0.1
|
2.4-10
|
22
|
|
|
|
|
|
|
|
NO3-
|
7
|
3
|
5.3±0.1
|
2.4-8.2
|
22
|
|
|
|
|
|
|
|
N0
|
6
|
4
|
5.7±0.1
|
2.2-8.2
|
18
|
|
|
|
|
|
|
DTF
|
NH4+
|
79
|
76
|
80.9±0.6
|
62.5-107
|
9
|
83
|
72
|
80.5±0.5
|
65.1-104.5
|
8
|
|
|
NO3-
|
80
|
76
|
82.7±0.6
|
66-130
|
9
|
87
|
88
|
87±0.4
|
74.3-98.1
|
6
|
|
|
N0
|
77
|
75
|
79.5±0.6
|
58-102
|
9
|
77
|
75
|
79.6±0.6
|
58.1-104.2
|
9
|
|
PH
|
NH4+
|
104
|
147
|
117.1±1.6
|
73.6-170.4
|
16
|
79
|
98
|
102.6±1
|
61.2-146.1
|
17
|
|
|
NO3-
|
91
|
136
|
108.1±1.3
|
72.4-157.9
|
14
|
87
|
88
|
86.2±1
|
57.2-122.7
|
16
|
|
|
N0
|
92
|
139
|
111.3±1.5
|
63.2-220.8
|
17
|
61
|
77
|
71.6±0.9
|
47.3-99.7
|
15
|
|
PL
|
NH4+
|
25
|
26
|
23.8±0.1
|
17.5-27.6
|
7
|
19
|
26
|
21.7±0.2
|
16.3-30.1
|
10
|
|
|
NO3-
|
24
|
26
|
23.2±.1
|
19.85-27.2
|
6
|
17
|
22
|
19.7±0.8
|
15.3-32.3
|
10
|
|
|
N0
|
24
|
25
|
23.4±0.1
|
18.5-26.5
|
7
|
18
|
18
|
18.1±0.1
|
14.2-26
|
10
|
|
FLL
|
NH4+
|
30
|
37
|
30.8±.04
|
21.8-44.5
|
15
|
26
|
28
|
29.4±0.5
|
20.5-55.5
|
18
|
|
|
NO3-
|
28
|
38
|
28.6±0.3
|
21.2-39.5
|
13
|
22
|
35
|
26.2±0.4
|
17.4-43.7
|
19
|
|
|
N0
|
27
|
36
|
28.2±0.3
|
20.1-38.4
|
14
|
14
|
17
|
17.9±0.3
|
8.25-28.1
|
17
|
|
FLW
|
NH4+
|
1.1
|
1.4
|
1.4±0.02
|
0.98-2
|
12.9
|
1.3
|
1.5
|
1.3±0.1
|
1.02-2
|
21
|
|
|
NO3-
|
1.2
|
1.8
|
1.3±.02
|
0.97-1.92
|
14.2
|
1.2
|
1.5
|
1.1±0.1
|
0.7-1.1
|
18
|
|
|
N0
|
1.2
|
1.2
|
1.2±0.01
|
1-2.6
|
16
|
0.8
|
1.1
|
1.00±0.02
|
0.7-2.3
|
20
|
|
FLA
|
NH4+
|
24
|
40
|
32.2±0.6
|
18.8-52.0
|
20
|
26
|
33
|
29.9±0.7
|
17.4-83.2
|
28
|
|
|
NO3-
|
25
|
50
|
27.2±0.5
|
16.1=50.4
|
21
|
20
|
38
|
22.7±0.6
|
9.1-62.7
|
32
|
|
|
N0
|
23
|
33
|
26.2±0.5
|
17.2-52.2
|
20
|
8
|
14
|
13.4±0.3
|
2.6-33.7
|
30
|
|
PLL
|
NH4+
|
37
|
51
|
44.3±0.6
|
30.2-62.3
|
15
|
40
|
43
|
42.5±0.6
|
29.2-65.6
|
18
|
|
|
NO3-
|
36
|
47
|
40.1±0.5
|
29.4-56.5
|
13
|
30
|
49
|
38.3±0.6
|
23.2-61.8
|
20
|
|
|
N0
|
36
|
41
|
40.2±0.5
|
28.7-56.9
|
13
|
22
|
29
|
28.4±0.4
|
20-40.4
|
17
|
|
PLW
|
NH4+
|
1
|
1.1
|
1.2±0.01
|
0.8-1.6
|
12
|
1.1
|
1.2
|
1.1±0.02
|
0.69-2.2
|
17
|
|
|
NO3-
|
1
|
1.2
|
1.0±0.01
|
0.7-1.3
|
12
|
0.9
|
1.1
|
0.9±0.02
|
0.57-1.9
|
22
|
|
|
N0
|
0.9
|
1.2
|
1.0±0.01
|
0.7-1.3
|
11
|
0.6
|
0.7
|
0.7±0.01
|
0.49-1.3
|
18
|
|
SL
|
NH4+
|
9
|
9
|
9.1±.04
|
7.5-10.2
|
6
|
9
|
8
|
9.2±0.05
|
7.7-10.6
|
6
|
|
|
NO3-
|
9
|
9
|
9.2±0.05
|
7.5-11.2
|
6
|
9
|
9
|
9.1±0.04
|
7.65-10.7
|
6
|
|
|
N0
|
10
|
8
|
9.1±0.04
|
7.4-10.3
|
6
|
10
|
8
|
9.1±0.04
|
7.4-10.3
|
6
|
|
SB
|
NH4+
|
2
|
3
|
2.5±.01
|
2-2.95
|
7
|
2
|
3
|
2.4±0.01
|
2.1-2.9
|
11
|
|
|
NO3-
|
2
|
3
|
2.6±0.01
|
2.1-3.1
|
7
|
2
|
3
|
3.5±0.01
|
2.1-3.2
|
7
|
|
|
N0
|
2
|
3
|
2.6±0.01
|
2.1-3.2
|
7
|
2
|
3
|
2.6±0.01
|
2.1-3.2
|
7
|
|
SLB
|
NH4+
|
4
|
3
|
3.7±.02
|
2.6-4.4
|
9
|
4
|
3
|
3.6±0.03
|
2.39-4.84
|
11
|
|
|
NO3-
|
4
|
3
|
3.6±.04
|
1.3-4.7
|
11
|
4
|
4
|
3.6±0.1
|
2.85-4.60
|
10
|
|
|
N0
|
4
|
3
|
3.6±0.03
|
2.5-6.1
|
12
|
4
|
3
|
3.5±0.1
|
2.58-4.62
|
10
|
|
SF
|
NH4+
|
71
|
52
|
74.8±1
|
49-89.1
|
18
|
85
|
86
|
80.9±0.7
|
55.2-96.3
|
11
|
|
|
NO3-
|
60
|
73
|
74.7±0.7
|
48.8-88.8
|
11
|
59
|
98
|
79.6±0.8
|
50.3-97.6
|
11
|
|
|
N0
|
72
|
80
|
71.1±1
|
17.2-90.1
|
18
|
65
|
78
|
79.6±1.1
|
30.2-95.4
|
17
|
|
SS
|
NH4+
|
29
|
48
|
31.3±1
|
7.0-86.8
|
40
|
15
|
14
|
19.1±0.8
|
3.7-44.7
|
47
|
|
|
NO3-
|
40
|
27
|
25.1±0.7
|
10.1-41.1
|
32
|
41
|
5
|
20.4±0.8
|
55.2-96.3
|
45
|
|
|
N0
|
28
|
20
|
28.1±1.07
|
9.8-82.7
|
45
|
35
|
22
|
20.4±1.1
|
4.6-69.8
|
66
|
|
GY=grain yield, BG= biological yield, HI= harvest
index, TT= total tillers, DTF= days to 50 % flowering, TT= total tiller, ET=
effective tiller, SB= seedling biomass, PH= plant height, PL=panicle length,
FLL=flag leaf length, FLW=flag leaf width, FLA=flag leaf area, PLL=
penultimate leaf length, FLW=flag leaf width, FLA=flag leaf area, PLL=
penultimate leaf length, PLW= penultimate leaf width, SL= seed length, SB=
seed breadth, SLB= seed length and breadth ratio, SF=Spikelet fertility.
|
|
Table 3. Mean phenotypic performance, range, standard deviation (SD),
Coefficient of variance (CV %) of investigated yield and yield related traits
of 32 selected RILs and their parents under differential N regimes and
environments during wet season 2015
|
|
|
Traits
|
N
|
IRRIGATED (I)
RILs
|
RAINFEED (R)
RILs
|
TERMINAL STAGE DROUGHT (TSD)
RILs
|
|
|
|
|
D
|
DD
|
Mean±SEm
|
Range
|
CV%
|
D
|
DD
|
Mean±SEm
|
Range
|
CV%
|
D
|
DD
|
Mean±SEm
|
Range
|
CV%
|
|
|
|
NH4+
|
316
|
224
|
284±17.1
|
117-522
|
34.1
|
80
|
321
|
136±8
|
48-261
|
36
|
113
|
135
|
130±8.6
|
20-260
|
37
|
|
|
GY
|
NO3-
|
292
|
241
|
233.3±8.9
|
135-381
|
22
|
71
|
239
|
125±10
|
29-248
|
46
|
62
|
103
|
90±4.6
|
37-177
|
29
|
|
|
|
N0
|
223
|
106
|
221.3±12.9
|
90.6-383
|
33
|
52
|
162
|
88±6
|
20-162
|
39
|
70
|
115
|
89±4.5
|
50-179
|
32
|
|
|
|
NH4+
|
640
|
594
|
849.9±41.0
|
372-1410
|
27.3
|
291
|
962
|
588±27
|
317-1042
|
26
|
439
|
497
|
528±11
|
398-665
|
12
|
|
|
BY
|
NO3-
|
631
|
494
|
674±31.3
|
427-1388
|
26
|
362
|
724
|
505±23
|
247-740
|
26
|
257
|
396
|
365±8.2
|
257-432
|
13
|
|
|
|
N0
|
514
|
221
|
613±26.1
|
310.4-921
|
24
|
208
|
518
|
375±15
|
151-546
|
23
|
271
|
279
|
327±8.3
|
255-424
|
15
|
|
|
|
NH4+
|
49
|
37
|
37±1.2
|
11.4-46.7
|
19.7
|
27
|
33
|
23±0.9
|
9.4-36.3
|
23
|
26
|
27
|
27±1.4
|
4-49
|
34
|
|
|
HI
|
NO3-
|
43
|
40
|
35.2±0.8
|
25.2-43.3
|
13
|
19
|
32
|
22±1.0
|
11.9-35
|
23
|
24
|
26
|
24±1.0
|
9.6-40
|
24
|
|
|
|
N0
|
42
|
36
|
35.5±1.2
|
19.2-40.3
|
19
|
25
|
30
|
21±1.0
|
13-33
|
25
|
26
|
36
|
23±1.3
|
5-38
|
22
|
|
|
|
NH4+
|
|
|
|
|
|
429
|
357
|
440±13
|
295-617
|
17
|
|
|
|
|
|
|
|
TT/m2
|
NO3-
|
|
|
|
|
|
362
|
724
|
394±7.7
|
139-301
|
19
|
|
|
|
|
|
|
|
|
N0
|
|
|
|
|
|
488
|
323
|
226±13
|
275-569
|
20
|
|
|
|
|
|
|
|
|
NH4+
|
9
|
5.3
|
8.1±0.28
|
5.0-12.0
|
19
|
|
|
|
|
|
12
|
6
|
9±0.2
|
6.3-12
|
14
|
|
|
TT/P
|
NO3-
|
7.5
|
3.6
|
6.76±0.2
|
3.6-9.5
|
19
|
|
|
|
|
|
10
|
5.8
|
8.6±0.1
|
5.8-11
|
13
|
|
|
|
N0
|
6.7
|
3.4
|
6.5±0.2
|
3.4-9.4
|
19
|
|
|
|
|
|
8.3
|
5.5
|
7.8±0.1
|
5.5-10
|
12
|
|
|
|
NH4+
|
8.5
|
5
|
6.9±0.3
|
3-9.9
|
25
|
|
|
|
|
|
12
|
6
|
8±0.2
|
5.6-11
|
16
|
|
|
ET/P
|
NO3-
|
7.3
|
3.6
|
6.2±0.2
|
3.6-9.3
|
19
|
|
|
|
|
|
8.5
|
6
|
7.8±0.1
|
5.9-9.7
|
13
|
|
|
|
N0
|
6.1
|
3.4
|
6.2±0.2
|
3.4-8.5
|
19
|
|
|
|
|
|
8
|
5.1
|
7.2±0.1
|
5.1-9.2
|
15
|
|
|
|
NH4+
|
2.2
|
4.2
|
3.85±0.28
|
2.0-9.3
|
33
|
5.8
|
6.5
|
5.0±0.2
|
2.4-7.9
|
26
|
|
|
|
|
|
|
|
SB
|
NO3-
|
1.3
|
3.5
|
2.9±0.10
|
1.3-3.5
|
28
|
2.9
|
5
|
3.4±0.1
|
2.0-6.1
|
31
|
|
|
|
|
|
|
|
|
N0
|
2.3 8
|
2.5 3
|
3.1±0.1
|
1.9-4.8
|
24
|
1.5
|
2.2
|
2.2±0.1
|
1.2-3.3
|
24
|
|
|
|
|
|
|
|
|
NH4+
|
80
|
70
|
76.4±1.4
|
66-102.5
|
11
|
79
|
67
|
76±1.6
|
62.5- 102.5
|
17
|
75
|
80
|
80±1.5
|
71-102
|
11
|
|
|
DTF
|
NO3-
|
79
|
70
|
78.4±1.5
|
67.5-104
|
10
|
79
|
67
|
76±1.6
|
65-104
|
11
|
77
|
78
|
81±1.4
|
71-103
|
10
|
|
|
|
N0
|
76
|
68
|
80.4±1.5
|
66.5-103
|
11
|
83
|
68
|
78±1.4
|
68-100
|
11
|
77
|
79
|
81±1.4
|
71-102
|
10
|
|
|
|
NH4+
|
79
|
128
|
106±3.7
|
74-147
|
20
|
81
|
130
|
99±3.2
|
73.6-135
|
12
|
76
|
128
|
101±3.3
|
73-141
|
18
|
|
|
PH
|
NO3-
|
75
|
116
|
97.7±3.3
|
64.6-132
|
19
|
77
|
122
|
92±3.1
|
64-125
|
19
|
70
|
128
|
90±2.9
|
66-129
|
19
|
|
|
|
N0
|
81
|
117
|
96.1±2.9
|
64.4-132
|
17
|
72
|
103
|
82±2.6
|
63-116
|
18
|
65
|
122
|
89±2.8
|
63-123
|
19
|
|
|
|
NH4+
|
22
|
23
|
23.4±0.4
|
20.3-32.6
|
10
|
20
|
25. 5
|
22±0.4
|
18.7-27.4
|
10
|
21
|
26
|
22±0.3
|
17.5-26
|
8
|
|
|
PL
|
NO3-
|
22
|
24
|
22.5±0.3
|
19.1-30.3
|
9
|
18
|
24
|
21±0.4
|
18-26.4
|
10
|
19
|
21
|
20±0.3
|
18-25
|
9
|
|
|
|
N0
|
21
|
22
|
21.6±0.3
|
18.3-25.8
|
7
|
17
|
20
|
19±0.3
|
14-34
|
9
|
20
|
19
|
20±0.4
|
16-26
|
10
|
|
|
|
NH4+
|
26
|
33
|
30.5±0.9
|
21.1-44.2
|
17
|
24
|
37
|
39±1.0
|
19-45
|
19
|
22
|
31
|
27±0.5
|
21-35
|
11
|
|
|
FLL
|
NO3-
|
24
|
30
|
26.6±0.6
|
20.1-35.3
|
14
|
28
|
45
|
32±1.2
|
22-52
|
20
|
19
|
25
|
24±0.5
|
19-36
|
14
|
|
|
|
N0
|
20
|
29
|
24.5±0.7
|
17.8-35.3
|
16
|
18
|
30
|
21±0.8
|
14.5-34
|
21
|
21
|
23
|
24±0.5
|
19-31
|
13
|
|
|
|
NH4+
|
1.1
|
1.5
|
1.3±0.1
|
1.06-1.72
|
13
|
1.4
|
2
|
1.4±0.1
|
1.2-2
|
14
|
1.2
|
1.7
|
1.4±0.03
|
0.8-1.7
|
13
|
|
|
FLW
|
NO3-
|
1.2
|
1.4
|
1.2±0.1
|
0.9-1.5
|
13
|
1.4
|
1.8
|
1.3±0.03
|
22.6-51.9
|
12
|
1.1
|
1.5
|
1.3±0.03
|
0.9-1.9
|
14
|
|
|
|
N0
|
0.9 4
|
1.4
|
0.9±0.1
|
0.6-1.3
|
19
|
1
|
1.6
|
1.2±0.1
|
0.8-2.1
|
11
|
1
|
1.6
|
1.3±0.03
|
0.8-1.7
|
12
|
|
|
|
NH4+
|
23
|
40
|
30.4±1.4
|
18.9-54.3
|
26
|
27
|
55
|
35.6-1.6
|
22-60
|
27
|
20
|
39
|
28.1±0.9
|
17-39
|
19
|
|
|
FLA
|
NO3-
|
22
|
33
|
23.8±0.8
|
16.9-36.7
|
20
|
31
|
63
|
34.5±1.9
|
21.1-67.6
|
31
|
16
|
28
|
24.9±0.8
|
16-39
|
19
|
|
|
|
N0
|
14.5
|
28.8
|
17.6±0.9
|
9.3-29.9
|
28
|
13
|
38
|
20.8±1.3
|
20.8-40.9
|
32
|
16
|
29
|
23±0.7
|
16-33
|
20
|
|
|
|
NH4+
|
33
|
48
|
38.8±1.2
|
29-60.2
|
18
|
38
|
58
|
41-1.1
|
31-58
|
16
|
32
|
40
|
37.6±0.7
|
28.9-47.9
|
11
|
|
|
PLL
|
NO3-
|
32
|
44
|
36.5±1.1
|
26.6-51.2
|
17
|
32
|
52
|
38.3±1.2
|
28-56
|
18
|
30
|
45
|
36.4±0.4
|
29.4-47.1
|
10
|
|
|
|
N0
|
27
|
43
|
34.5±1.2
|
25.3-51.2
|
19
|
26
|
47
|
32.5±1.1
|
23.2-47.4
|
19
|
35
|
43
|
36±0.7
|
28-46
|
12
|
|
|
|
NH4+
|
0.8
|
1.4
|
1.07±0.1
|
0.51-1.44
|
16
|
1.1
|
1.6
|
1.2±0.1
|
0.9-3.1
|
17
|
0.9
|
1.3
|
1.1±0.03
|
0.6-1.4
|
17
|
|
|
PLW
|
NO3-
|
1.07
|
1.1
|
1.0±0.1
|
0.7-1.3
|
14
|
1.1
|
1.5
|
1.1±0.02
|
0.8-1.6
|
14
|
0.7
|
1.3
|
0.9±0.03
|
0.4-1.5
|
22
|
|
|
|
N0
|
0.94
|
1.49
|
1.0±0.1
|
0.6-1.5
|
19
|
0.9
|
1.2
|
0.97±0.1
|
0.6-1.3
|
17
|
0.6
|
1.2
|
0.9±0.03
|
0.63-1.36
|
21
|
|
|
|
NH4+
|
9.3
|
9.6
|
9.1±0.1
|
7.2-10
|
7
|
8.9
|
8.7
|
8.8±0.1
|
7.2-10.2
|
7
|
|
|
|
|
|
|
|
SL
|
NO3-
|
8.7
|
9.7
|
9.0±0.1
|
7.3-9.9
|
6
|
8.8
|
8.9
|
8.7±0.01
|
7.8-9.2
|
3
|
|
|
|
|
|
|
|
|
N0
|
8.1
|
8.6
|
8.9±0.1
|
7.4-10.1
|
7
|
9
|
8.7
|
8.5±0.1
|
7.4-9.8
|
7
|
|
|
|
|
|
|
|
|
NH4+
|
2.6
|
2.7
|
2.6±0.1
|
2.3-2.9
|
6
|
2.4
|
2.5
|
2.5±0.02
|
2.2-2.7
|
5
|
|
|
|
|
|
|
|
SB
|
NO3-
|
2.4
|
2.5
|
2.5±0.1
|
2.3-2.8
|
5
|
2.3
|
2.4
|
2.5±0.02
|
2.2-2.6
|
5
|
|
|
|
|
|
|
|
|
N0
|
2.5
|
2.7
|
2.6±0.1
|
2.3-2.8
|
4
|
2.4
|
2.7
|
2.5±0.03
|
2.1-2.9
|
7
|
|
|
|
|
|
|
|
|
NH4+
|
3.6
|
3.5
|
3.4±0.1
|
3.1-4.1
|
7
|
3.6
|
3.4
|
3.5±0.0
|
3.1-4.1
|
7
|
|
|
|
|
|
|
|
SLB
|
NO3-
|
3.5
|
3.8
|
3.5±0.1
|
2.9-4.1
|
8
|
3.7
|
3.6
|
3.5±0.03
|
3.2-4.0
|
5
|
|
|
|
|
|
|
|
|
N0
|
3.2
|
3.1
|
3.5±0.1
|
3.1-4.1
|
7
|
3.7
|
3.1
|
3.4±0.05
|
2.5-4.0
|
10
|
|
|
|
|
|
|
|
|
NH4+
|
2.3
|
2.8
|
2.6±0.1
|
1.8-3.1
|
11
|
2
|
2.5
|
2.3±0.05
|
1.7-3
|
12
|
|
|
|
|
|
|
|
1000W
|
NO3-
|
2.3
|
2.6
|
2.6±0.5
|
1.7-3.0
|
13
|
1.8
|
2.5
|
2.4±0.01
|
1.6-3
|
13
|
|
|
|
|
|
|
|
|
N0
|
2.3
|
2.4
|
2.5±0.1
|
1.7-3.0
|
12
|
2.1
|
3
|
2.4±0.1
|
1.7-3.1
|
14
|
|
|
|
|
|
|
|
GY=grain yield,
BG= biological yield, HI= harvest index, TT= total tillers, DTF= days to 50 %
flowering, TT= total tiller, ET= effective tiller, SB= seedling biomass, CGR=
crop growth rate, PH= plant height, PL=panicle length, FLL=flag leaf length,
FLW=flag leaf width, FLA=flag leaf area, PLL= penultimate leaf length, ,
FLW=flag leaf width, FLA=flag leaf area, PLL= penultimate leaf length, , PLW=
penultimate leaf width, SL= seed length, SB= seed breadth, SLB= seed length
and breadth ratio, TT/P= total tiller per plant, ETP= Effective tillers per
plant, SB= Seedling biomass.
|
|
|
Table 4. Mean phenotypic performance, range, standard deviation (SD), Coefficient
of variance (CV %) of investigated chlorophyll parameters, NUE and its
component traits of 32 selected RILs and their parents under differential N
regimes and environments during wet season, 2015.
|
|
|
|
Source of variation
|
IRRIGATED (I)
RILs
|
RAINFED (R)
RILs
|
|
Traits
|
N
|
G TMSS DF=31
|
N TMSS DF=2
|
E TMSS DF=1
|
EXG TMSS
DF=31
|
NXG
TMSS DF=62
|
EXN
TMSS DF=2
|
EXNXG
TMSS DF=62
|
D
|
DD
|
Mean±SEm
|
Range
|
CV%
|
D
|
DD
|
Mean±SEm
|
Range
|
CV%
|
|
|
NH4+
|
|
|
|
|
|
|
|
35
|
32
|
32±0.3
|
29-38
|
6
|
35
|
31
|
32±0.4
|
29-38
|
6
|
|
SPAD till
|
NO3-
|
49.2**
|
186**
|
NS
|
NS
|
5.3 *
|
NS
|
NS
|
33
|
30
|
31±0.4
|
26-37
|
7
|
33
|
30
|
31±0.4
|
26-36
|
7
|
|
|
N0
|
|
|
|
|
|
|
|
31
|
31
|
30±0.4
|
26-35
|
7
|
32
|
30
|
30±0.4
|
26-34
|
7
|
|
|
NH4+
|
|
|
|
|
|
|
|
37
|
35
|
36±0.4
|
30-42
|
6
|
38
|
40
|
37±0.6
|
28-43
|
8
|
|
SPAD now
|
NO3-
|
50.2**
|
190**
|
NS
|
NS
|
6.6*
|
NS
|
NS
|
36
|
32
|
35±1.3
|
28-41
|
6
|
34
|
37
|
35±0.1
|
29-41
|
8
|
|
|
N0
|
|
|
|
|
|
|
|
32
|
29
|
32±0.5
|
24-37
|
7
|
31
|
28
|
28±0.5
|
22-33
|
10
|
|
|
NH4+
|
|
|
|
|
|
|
|
1.4
|
1.2
|
1.2±0.1
|
1-1.4
|
10
|
1.7
|
1.6
|
1.5±0.1
|
1.1-2
|
12
|
|
GNC
|
NO3-
|
0.05**
|
1.6**
|
3.1**
|
0.04**
|
0.03**
|
0.4**
|
0.29 *
|
1.1
|
1.2
|
1.1±0.3
|
0.9-1
|
8
|
1.2
|
1.1
|
1.3±0.2
|
1.1-2
|
11
|
|
|
N0
|
|
|
|
|
|
|
|
1.0
|
1.2
|
1.1±0.2
|
0.7-1
|
7
|
1.2
|
1.1
|
1.2±0.1
|
1.0-2
|
8
|
|
|
NH4+
|
|
|
|
|
|
|
|
8.2
|
6.9
|
7.3±0.1
|
6.1-8.7
|
10
|
10
|
9.6
|
8.9±0.2
|
6.9-11
|
13
|
|
GPC
|
NO3-
|
1.8**
|
59**
|
114 **
|
1.6**
|
1.0**
|
15.4 **
|
1.0**
|
6.5
|
7.4
|
6.8± 0.1
|
5.8-8.1
|
8
|
7.5
|
6.9
|
8.2±0.2
|
6.8-10
|
12
|
|
|
N0
|
|
|
|
|
|
|
|
5.8
|
7.0
|
6.6±0.1
|
4.6-7.8
|
8
|
7.6
|
6.9
|
6.9±0.1
|
6.1-8
|
8
|
|
|
NH4+
|
|
|
|
|
|
|
|
0.6
|
0.5
|
0.5±0.1
|
0.3-0.6
|
18
|
1.1
|
0.7
|
0.9±0.1
|
0.6-2
|
21
|
|
SNC
|
NO3-
|
0.09**
|
0.89**
|
9.3**
|
0.04**
|
0.02**
|
0.47**
|
0.01**
|
0.5
|
0.5
|
0.47±0.1
|
0.3-0.6
|
12
|
0.9
|
0.5
|
0.8±0.1
|
0.5-1
|
19
|
|
|
N0
|
|
|
|
|
|
|
|
0.5
|
0.5
|
0.45±0.0
|
0.2-0.6
|
14
|
0.7
|
0.5
|
0.6±0.1
|
0.4-1
|
21
|
|
|
NH4+
|
|
|
|
|
|
|
|
2.4
|
3.0
|
3.5±0.2
|
1.4-6.9
|
35
|
1.4
|
3.6
|
2.0±0.1
|
0.8-3
|
37
|
|
GNY
|
NO3-
|
3.0**
|
35**
|
158**
|
2.1**
|
0.73**
|
2.73**
|
0.61**
|
2.8
|
2.9
|
2.6±0.1
|
1.6-4.8
|
23
|
0.9
|
2.7
|
1.7±0.1
|
0.5-3
|
44
|
|
|
N0
|
|
|
|
|
|
|
|
2.6
|
2.4
|
2.4±0.1
|
0.9-4.1
|
22
|
0.8
|
1.9
|
1.0±0.1
|
0.2-2
|
21
|
|
|
NH4+
|
1.6**
|
84**
|
53**
|
1.6**
|
0.9**
|
13**
|
0.7**
|
1.5
|
2.1
|
2.7±0.2
|
1.4-4.8
|
33
|
2.6
|
6.1
|
4.0±0.1
|
2.5-7
|
23
|
|
SNY
|
NO3-
|
|
|
|
|
|
|
|
2.0
|
1.9
|
2.1±0.2
|
1.1-5.3
|
35
|
2.8
|
2.6
|
3.0±0.1
|
1.9-5
|
21
|
|
|
N0
|
|
|
|
|
|
|
|
1.9
|
1.5
|
1.7±0.1
|
0.5-3.0
|
32
|
1.4
|
1.8
|
1.7±0.1
|
0.8-3
|
41
|
|
|
NH4+
|
6.3**
|
22**
|
27**
|
5.6**
|
2.3**
|
17.7**
|
1.7**
|
3.9
|
5.1
|
6.3±0.3
|
2.9-11
|
28
|
4.0
|
9.6
|
6.1±0.2
|
3.9-10
|
23
|
|
BNY
|
NO3-
|
|
|
|
|
|
|
|
4.7
|
4.8
|
4.7±0.1
|
2.8-10
|
26
|
3.7
|
5.4
|
4.7±0.2
|
2.6-7
|
25
|
|
|
N0
|
|
|
|
|
|
|
|
4.5
|
3.9
|
4.2±0.2
|
2.0-6.3
|
24
|
2.2
|
3.7
|
2.8±0.1
|
1.1-4.3
|
27
|
|
|
NH4+
|
381**
|
121*
|
45,655**
|
183**
|
84**
|
20**
|
84**
|
58
|
58
|
57±1.6
|
25-69
|
17
|
35
|
36
|
35±1.2
|
17-45
|
21
|
|
NHI
|
NO3-
|
|
|
|
|
|
|
|
57
|
60
|
56±1.0
|
45-66
|
10
|
24
|
50
|
34±1.7
|
16-50
|
27
|
|
|
N0
|
|
|
|
|
|
|
|
57
|
61
|
56.7±1.6
|
36 ±74
|
11
|
36
|
49
|
32±1.7
|
20-52
|
24
|
|
|
NH4+
|
0.25**
|
0.17**
|
0.01**
|
0.01**
|
0.003**
|
0.11**
|
0.003*
|
0.1
|
0.2
|
0.20±0.1
|
0.1-0.3
|
28
|
0.1
|
0.4
|
0.22±0.1
|
0.1-0.3
|
24
|
|
NUpE
|
NO3-
|
|
|
|
|
|
|
|
0.2
|
0.2
|
0.18±0.0
|
0.1-0.3
|
26
|
0.1
|
0.2
|
0.17±0.1
|
0.1-0.2
|
25
|
|
|
N0
|
|
|
|
|
|
|
|
0.2
|
0.2
|
0.15±0.0
|
0.1-0.3
|
23
|
0.09
|
0.1
|
0.10±0.2
|
0.04-0.2
|
28
|
|
|
NH4+
|
343**
|
1,753**
|
47,295**
|
166**
|
100**
|
NS
|
104**
|
42.8
|
50.3
|
45±1.4
|
18-59
|
18
|
20
|
22
|
21.8±0.8
|
9.4-33
|
22
|
|
NUtE
|
NO3-
|
|
|
|
|
|
|
|
53.3
|
48.9
|
49±1.1
|
34-60
|
13
|
19
|
43
|
25.1±1.4
|
9.5-43
|
31
|
|
|
N0
|
|
|
|
|
|
|
|
60.3
|
52.6
|
51±1.7
|
28-66
|
12
|
28
|
42
|
30.5±1.5
|
18-45
|
27
|
|
|
NH4+
|
|
|
|
|
|
|
|
5.6
|
8.5
|
9.8±0.5
|
3.8-17
|
34
|
3.1
|
8.4
|
5.0±0.3
|
1.8-9
|
35
|
|
NUE
|
NO3-
|
29.5**
|
33**
|
2,003**
|
16.5**
|
6.5**
|
99**
|
4.8 **
|
8.5
|
7.8
|
9.3±0.3
|
4.5-12
|
21
|
2.7
|
9.0
|
4.7±0.4
|
1.1-9.9
|
46
|
|
|
N0
|
|
|
|
|
|
|
|
12.0
|
9.0
|
8.8±0.5
|
4.0-17
|
20
|
2.4
|
6.1
|
3.3±0.2
|
0.7-6.1
|
39
|
|
** significance at .05, .01 levels
respectively, SPADtill = SPAD at tillering , SPADflow = SPAD at flowering,
GNC= grain nitrogen content (%), GPC= grain protein content (%),SNC= straw N
content (SNC), GNY = grain N yield (g/m2), SNY = straw nitrogen yield (g/m2),
BNY= biological N yield (g/m2), NHI = nitrogen harvest index (%), NUpE=
nitrogen uptake efficiency (gg-1 N), NUtE= nitrogen utilization efficiency
((gg-1 N) , NUE= nitrogen use efficiency ((gg-1 N).
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
QTL analysis
A total of 58 QTLs conferring the corresponding five
traits were detected under three N forms and two environments (table 4); that
as a matter of fact included 14, 15 & 11 QTLs under NH4+, NO3- and N0
level of irrigated condition and 5, 10 & 1 QTLs under NH4+, NO3- and N0
level of rainfed conditions respectively. These QTLs were mapped to different
genomic regions of all rice chromosomes and most of them were on chromosomes 1
and 9.
Expression of GS and GOGAT gene
families under different N forms
Plant GS and GOGAT genes are very important in N
assimilation, but the systematic expression patterns of the GS and GOGAT genes
families have not yet been clearly established. Thus, we examined the level of
expression of each member of the GS and GOGAT gene families under NH4+ and N03- treatment using
quantitative real time PCR.
Actin Vs GS gene family
GS1 and GS2 are two major isoforms of
Glutamine Synthetase (GS). GS gene family comprises of three cytosolic GS1 gene
(OsGln1; 1, OsGln1;2 and OsGln1;3) and a single plastidic
OsGln2 gene. Distinct roles for this enzyme have been suggested by a
number of studies on organ, tissue and development (Harrison et al.,
2000; Weber and Harrison, 2002).
In the present qPCR experiment for GS
genes, the levels of transcripts in organs under study were calculated relative
to the Actin gene. The relative expression values more than one shows the
increased expression or up regulation while values less than one shows the
decreased expression or down regulation as compared to control. Under both NH4+ and NO3- treatment,
two of the genes, OsGln1;1 & OsGln1;2 showed a markedly
preferential expression pattern in roots whereas OsGln1;2 was mainly
expressed in shoot. OsGln2 showed the differential expression level both
in root and shoot tissues among all selected genotypes. Furthermore,
significant effect of NO3- and NH4+ treatment was observed on GS
expression. Overall, in root tissue, OsGln1;1 gene seems to be
upregulated in root under both NH4+ and NO3 - treatment with line no.10 (14.82)
manifesting significantly strong expression while in shoot line no. 4 (55.13)
and line no. 3 (6.16) showed strong expression under NH4+ and NO3-
treatment. OsGln1;2 in root showed significant down-regulation by NH4+ treatment except for line no. 10
(8.1)and up-regulation by NO3- with strong expression harbouring in line no.4
(13.08) while in shoot, excluding line no. 9 (3.27) a significant down
regulation and weak expression was observed by NH4+ treatment while NO3-
treatment resulted in up regulation as well as down regulation in 10 selected
rice genotypes. OsGln1;3 gene in root exhibited strong expression in
line no. 4 (10.12) & 5 (7.54) while in shoot most of the genes were
up-regulated as a result of NH4+ treatment. Additionally, most of the genotypes showed
significant up regulation of OsGln1;3 by NO3-
treatment in both root and shoot with line no.3 (11.91) manifesting strong
expression. NH4+ treatment revealed strong expression of OsGln2
gene in shoot of line no. 4 (125.8) and in root of line no. 1 (30.59) while in
NO3-treatment strong expression in shoot is visible in
line no. 4 (186.7) and line no. 10 (63.11) in root. This study suggest that OsGln1;
1, OsGln1;2 and OsGln1;3 shows reciprocal responses to NH4+ and NO3- supply
as oppose to proposed mechanism. Collectively, among all isoforms, OsGln2
which predominantly functions in green tissue and is indispensable for
assimilation of photo respiratory ammonia in high NUE genotypes revealed shoot
preferential expression pattern. OsGln2 was highly upregulated in line
no. 4 among all isoforms under both the N supply. This line is high yielding
and falls in dark green spectrum of colour classes which denotes the
relationship of GS in green tissues with grain yield.
Actin Vs GOGAT gene family
GOGAT catalyzes reductive transfer of the amide group of
Gln to 2-oxogluarate to form two Glu molecules. In rice plants, NADH-GOGAT
is coded by two genes, OsNADH-GOGAT1 (OsGlt1) and OsNADH-GOGAT2 (OsGlt2)
(Tabuchi et al., 2007b).
In the present qPCR experiment for GS
genes, the levels of transcripts in organs under study were calculated relative
to the Actin gene. The relative expression values more than one shows the
increased expression or up regulation while values less than one shows the
decreased expression or down regulation as compared to control. Expression of
OsGlt1 and OsGlt2 genes was strongly repressed by NH4+ and NO3- treatment for most
of the genotypes. Exceptionally, OsGlt1 gene in root tissue of line no.
7 (7.31) of green colour and line no. 10 (1.17) of yellow colour was
upregulated by NH4+ treatment while line no.4 (2.74) and line no.5 (2.50)
which falls in dark green and yellow colour in colour class was upregulated by
NO3- treatment. OsGlt1 gene in shoot tissue of
line no. 4 (1.46) showed increased expression in NH4+ treatment and line no. 9(2.71) of dark
green colour class and line no. 10 (2.35) of yellow colour class in NO3-
treatment. OsGlt2 gene
|
Table 5. Results of QTLs for five traits under three N
levels under two environments
|
|
Trait
|
|
|
Left marker
|
Right marker
|
LOD
|
Add.
|
R2
(%)
|
|
Grain Yield (GY)
1
|
|
1
|
RM3825
|
RM302
|
2.8
|
43.6
|
9.8
|
|
|
NH4+ I
|
7
|
HvSSR7-53
|
RM-2
|
3.05
|
-72.12
|
18.28
|
|
|
|
11
|
HvSSR11-2
|
HvSSR11-3
|
2.82
|
46.53
|
8.81
|
|
|
|
12
|
RM277
|
RM260
|
3.13
|
29.31
|
8.10
|
|
|
|
9
|
RM434
|
RM410
|
3.11
|
31.84
|
10.97
|
|
|
NO3- I
|
1
|
RM410
|
RM108
|
3.03
|
33.75
|
9.17
|
|
|
|
11
|
HvSSR11-2
|
HvSSR11-3
|
3.19
|
30.51
|
9.47
|
|
|
N0 I
|
1
|
RM449
|
RM-5
|
2.51
|
35.4
|
7.20
|
|
|
|
9
|
RM434
|
RM410
|
2.55
|
38.77
|
8.48
|
|
|
NH4+ R
|
1
|
RM449
|
RM5
|
3.42
|
-32.73
|
12.42
|
|
|
|
1
|
RM572
|
RM24
|
2.91
|
-38.10
|
14.31
|
|
|
|
1
|
HvSSR1-34
|
HvSSR1-49
|
2.56
|
-28.92
|
3.68
|
|
|
NO3- R
|
9
|
HvSSR9-25
|
HvSSR9-27
|
5.54
|
-35.88
|
11.70
|
|
|
|
9
|
RM434
|
RM410
|
3.97
|
-35.63
|
11.75
|
|
|
|
11
|
RM21
|
RM26334
|
3.38
|
-38.13
|
7.32
|
|
Biological Yield (BY)
|
|
|
|
|
|
|
|
|
|
NH +
|
12
|
HvSSR12-36
|
HvSSR12-40
|
3.85
|
-103.52
|
12.3
|
|
|
|
12
|
RM260
|
RM519
|
4.89
|
132.91
|
17.92
|
|
|
NO3- I
|
1
|
RM243
|
RM572
|
3.34
|
65.14
|
15.65
|
|
|
|
1
|
RM449
|
RM5
|
3.31
|
51.86
|
9.63
|
|
|
N0 I
|
6
|
RM136
|
RM340
|
3.78
|
-65.42
|
13.29
|
|
|
|
9
|
RM434
|
RM410
|
2.54
|
46.20
|
7.64
|
|
|
NO3- R
|
9
|
HvSSR9-25
|
HvSSR9-27
|
2.89
|
-58.03
|
8.02
|
|
|
|
9
|
HvSSR9-37
|
HvSSR9-57
|
3.88
|
-67.75
|
11.86
|
|
Harvest index (HI)
|
N0 R
|
7
|
RM2
|
RM11
|
2.57
|
31.22
|
9.15
|
|
|
|
1
|
HvSSR1-87
|
HvSSR1-89
|
2.7
|
3.39
|
8.02
|
|
|
NH4+ I
|
2
|
HvSSR2-27
|
HvSSR2-78
|
2.59
|
-3.77
|
7.91
|
|
|
|
4
|
HvSSR4-26
|
HvSSR4-35
|
3.12
|
3.64
|
9.1
|
|
|
|
1
|
RM1
|
HvSSR1-80
|
2.58
|
3.42
|
14.53
|
|
|
|
1
|
HvSSR1-89
|
RM259
|
3.21
|
3.23
|
11.52
|
|
|
NO3- I
|
9
|
RM434
|
RM410
|
2.73
|
3.03
|
12.1
|
|
|
|
9
|
RM410
|
RM108
|
2.81
|
2.72
|
9.98
|
|
|
|
11
|
HvSSR11-2
|
HvSSR11-3
|
3.93
|
3.27
|
13.44
|
|
|
|
12
|
RM20
|
HvSSR11-35
|
3.56
|
3.89
|
19.02
|
|
|
N0 I
|
1
|
RM486
|
RM14
|
5.13
|
5.71
|
31.5
|
|
|
|
3
|
HvSSR3-6
|
HvSSR3-9
|
3.92
|
4.46
|
17.05
|
|
|
|
9
|
RM434
|
RM410
|
3.22
|
2.94
|
8.68
|
|
|
|
11
|
RM206
|
RM254
|
2.57
|
3.54
|
10.49
|
|
|
NH4+ R
|
1
|
RM449
|
RM5
|
4.68
|
-3.57
|
19.83
|
|
|
|
12
|
RM270
|
RM17
|
2.53
|
-2.57
|
9.3
|
|
|
NO3-
R
|
1
|
RM449
|
RM5
|
3.43
|
-2.89
|
12.85
|
|
Chlorophyll content (CC)
|
|
|
|
|
|
|
|
|
|
NH4+ I
|
1
|
HvSSR1-87
|
HvSSR1-89
|
8.48
|
2.14
|
25.56
|
|
|
NO3-
I
|
1
|
HvSSR1-87
|
HvSSR1-89
|
2.83
|
1.3
|
8.27
|
|
|
|
1
|
RM572
|
RM24
|
4.27
|
-1.37
|
15.31
|
|
|
N0 I
|
1
|
HvSSR1-87
|
HvSSR1-89
|
8.8
|
1.89
|
27.34
|
|
|
|
9
|
RM288
|
RM253
|
2.58
|
0.98
|
6.73
|
|
Grain yield response
(GYR)
|
|
|
|
|
|
|
|
|
|
NH4+ I
|
8
|
HvSSR8-29
|
RM310
|
3.05
|
-41.28
|
15.57
|
|
|
|
8
|
RM72
|
RM515
|
3.27
|
-44.14
|
18.71
|
|
|
NO3-
I
|
11
|
RM202
|
RM229
|
2.89
|
-31.05
|
13.19
|
|
|
NO3- R
|
1
|
HvSSR1-34
|
HvSSR1-49
|
2.64
|
-23.71
|
5.07
|
|
|
|
9
|
HvSSR9-25
|
HvSSR9-27
|
7.35
|
-34.98
|
16.34
|
|
Biological yield response
|
|
|
|
|
|
|
|
|
(BYR)
|
NH4+ I
|
3
|
RM135
|
RM55
|
2.83
|
66.06
|
10.99
|
|
|
|
5
|
HvSSR5-66
|
RM163
|
2.51
|
-116.23
|
35.61
|
|
|
NO3- I
|
8
|
RM230
|
RM433
|
2.85
|
41.26
|
7.97
|
|
|
|
10
|
RM222
|
HvSSR10-34
|
2.76
|
-46.01
|
9.46
|
|
|
NH4+
R
|
6
|
HvSSR6-35
|
HvSSR6-44
|
3.41
|
-91.51
|
18.51
|
in root tissue of line no. 10 (3.81)
and in shoot tissue of line no. 4 (8.63) was upregulated by NH4+ treatment whereas line no. 3 (5.83)
showed significant upregulation in shoot tissue and line no. 9 (4.12) showed
significant upregulation in root tissue by NO3-
treatment.
The expression of these genes encoding
NADH-GOGAT remarkably reduced by external N forms and conditions. Our results
also showed that transcription of OsGlt1 is decreased in root and shoot
by N concentration while transcription of OsGlt2 is relatively increased
in root and shoot by N concentration. When N level is relatively high in soil,
expression of OsGlt1 and OsGlt2 can be decreased in order to
limit N acquisition but in the opposite side, expression of OsGlt1 and
OsGlt2 can be enhanced in order to increase N acquisition. This may be
kind of buffering effects in higher plants.
Expression of AMT and NRT transporters under different N
forms
Actin Vs AMT gene family
Although functionally not well characterized, twelve
putative AMT proteins have been identified located on different chromosome and
grouped in to five subfamilies (AMT1-AMT5) with one to three gene members (Deng
et al., 2007b; Lie et al., 2009b). So far, studies on expressions
and regulations of AMT genes in rice have been focused on the three genes of OsAMT1
family, which displayed different spatio-temporal expression patterns in
response to changes in N levels. OsAMT1;1, OsAMT1;2, OsAMT1;3
have been identified as members of AMT1, each showing a distinct expression
pattern: OsAMT1;1 shows constitutive expression in both shoots and roots
(Ding et al., 2011b). In the present study, similar results were
obtained influx of NH4+ ion resulted in up regulation and significant strong
expression of OsAMT 1.1 in both root and shoot tissue of line no. 5
(1.11, 10.52), 7 (1.82, 6.36) and line no. 9 (3.95, 4.00) whereas NO3-
influx resulted in strong expression in shoot tissue of line no. 3 (28.54). OsAMT1;
2 shows root-specific and ammonium-inducible expression (Ding et al.,
2011c). This is in contrary with present results in which up regulation of OsAMT1;2
was obtained in both root and shoot by NH4+ and NO3 - treatment of some
genotypes with exceptionally high and strong expression by NH4+ in shoot of line no. 4 (133.43) and in
shoot of line no. 3 (99.04) &5 (86.82) by NO3-.
Furthermore, NH4+ and NO3-treatment resulted in up
regulation of OsAMT1; 3 in both root and shoot of genotypes under study
with comparatively strongest expression in root and shoot tissue of line no. 9
(42.23) and line no. 4 (11.39) by NH4+ treatment and root & shoot tissues
of line no. 8 (11.63) and line no. 3 (11.55) by NO3-
treatment. In, contrary, Sonoda et al. (2003b) reported that OsAMT1;
3 shows root-specific and nitrogen-suppressible expression.
Our results dictates that OsAMT1;2
shows maximum mRNA accumulation in line no. 4 which also pertains high GS2
activity thus OsAMT1;2 expression increases with increase in endogenous
glutamine. It also seems that OsAMT1;2 functions in ammonium uptake in
ammonium enriched soils as its expression was highest in NH4+ treatment. These findings collaborate
with Sonoda et al. (2003c) who studied feedback regulation.
Actin Vs NRT gene family
When breeding crops that utilize nitrogen efficiently, it
is important to reveal the regulation of nitrate uptake at molecular level. We
focussed on expression of two nitrate uptake related genes, OsNRT2.4 and
OsNRT7.8, who are nitrate inducible and is mainly expressed in
parenchyma cells around the xylem. OsNRT2.4 and OsNRT7.8 mainly
participate in unloading nitrate from the xylem and maintains root-to-shoot
nitrate transport and vascular development (Xia et al., 2015). In the
present study, (Fig.3& table 5) significant upregulation of OsNRT2.4
was observed in shoot tissue of line no. 4 (6.10) and root tissue of line no. 5
(1.39) by NH4+ treatment while in shoot tissue of line no. 3 (10.26)
and root tissue of line no. 5 (3.4) by NO3- treatment. OsNRT7.8
revealed significant strong upregulation in root tissues of line no. 10 under NH4+ (19.35) and NO3-
(6.96) treatment.
Since rice roots are exposed to NO3-
nutrition under aerobic or water deficit condition and the importance of NO3-
nutrition can be dictated and the regulation & function of nitrate
transporter genes in rice are worthy of investigation. Strong upregulation of
OsNRT7.8 in line no. 10 which is actually of pale-yellow colour allow us
to select this line for breeding and production purposes for efficient nitrate
uptake under drought and water limited condition where major form of available
nitrogen is NO3-
Discussion
Zia et al. (2001) on nitrogen dynamics under
aerobic and anaerobic soil conditions observed the maximum drop in NH4+ concentration under aerobic
environment while high increase in NO3- concentration was
registered under aerobic condition. Chaturvedi et al. (2005) reported
earlier that more number of tillers in experiment might be due to the more
availability of nitrogen, which played a vital role in cell division.
Similarly, it was reported that the maximum number of tillers were at N200
level and the minimum at N0 level by Meena et al. (2003). Also, Fageria and Baligar (2001)
reported a quadratic relationship between rice grain yield and dry
matter yield of shoot. Woldeyessus et al. (2004) dictated the importance
of NUE varieties in future breeding program. Earlier reports by Tabuchi et
al., 2007; Martin et al., 2006 etc illustrates variation in enzyme
activity in genotypes and their role in enhancing NUE of a genotypes. In
the present study, the QTLs for grain yield, biological yield, harvest index,
chlorophyll content, grain yield response and biological yield response under
different nitrogen and water regimes have been speculated and identified. These
results are consistent with previous studies. Cho et al. (2007) found
that QTLs detected under high and low N levels are widely different. However,
the study of Tong et al. (2006) showed the presence of the same and
specially expressed QTLs under low and high N levels. Hu et al. (2012)
detected QTLs for N content and NUE in adjacent regions, respectively. Wei et
al. (2012) detected QTLs for nitrogen deficiency and nitrogen use
efficiency traits. For NDT and NUE traits, seven and eight QTLs were identified
in 2006 and 2007, respectively. Tong et al. (2011) analyzed the QTLs for
rice yield and its components under high, middle and low N levels, and detected
15, 23 and 19 QTLs at three N levels, respectively, thereby indicating the
occurrence of obvious interactions between QTLs for yield traits and N levels.
QTL for chlorophyll content (CC) was rarely reported previously. The genomic
region RZ599-RM53 on chromosome 2, where grain yield and biological response
QTL was located, was reported to have QTLs for grain yield under low nitrogen
and normal nitrogen by Wei et al. (2011). Rice plant have developed
intrinsic mechanism for uneven N distribution thus assimilate N efficiently in
roots by this kind of flexible and reciprocal regulatory mechanism, and control
rice growth and development. Similar results for OsGln1; 1, OsGln1;2,
OsGln1;3 and OsGln2 was earlier reported by Ishimaya et al.
(2004). Previous studies by Watanabe et al., (1996) show similar
responses and contradict previous report showing ammonium inducibility (Tobin et
al., 2001). Aaraki and Hasegawa (2006) revealed the importance of nitrate
uptake and nitrate transporters studies in rice. Systemic expression pattern
for nitrate transporters is not studied in past thus we open new insight for
nitrate-nitrogen studies.
Acknowledgment
I would like to extend my appreciation to Proff. Dr. S.B. Verulkar for his
guidance during the course of my research work. Without his valuable
assistance, this work would have not been completed. I thankfully acknowledge
the Department of Plant Molecular Biology and Biotechnology, IGKV, Raipur for
providing all necessary assistance and inputs required to implement this
research ideas. Finally, I am deeply indebted to Department of Science and
Technology, Government of India, for their cooperation and financial support in
form of Inspire fellowship.
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