high temperature at grain-filling stage affects nitrogen ... · from ammonia. glutamine synthetase...
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Rice Science, 2011, 18(3): 210−216 Copyright © 2011, China National Rice Research Institute Published by Elsevier BV. All rights reserved
High Temperature at Grain-filling Stage Affects Nitrogen Metabolism Enzyme Activities in Grains and Grain Nutritional Quality in Rice
LIANG Cheng-gang, CHEN Li-ping, WANG Yan, LIU Jia, XU Guang-li, LI Tian (Agronomy College, Sichuan Agricultural University, Chengdu 611130, China)
Abstract: Rice plants would more frequently suffer from high temperature (HT) stress at the grain-filling stage in future. A
japonica rice variety Koshihikari and an indica rice variety IR72 were used to study the effect of high temperature on dynamic
changes of glutamine synthetase (GS) activity, glutamate synthase (GOGAT) activity, glutamic oxalo-acetic transminase
(GOT) activity, glutamate pyruvate transminase (GPT) activity in grains and grain nutritional quality at the grain-filling stage.
Under HT, the activities of GOGAT, GOT, GPT and soluble protein content in grains significantly increased, whereas GS
activity significantly decreased at the grain-filling stage. In addition to the increase of protein and amino acids contents, it was
suggested that GOGAT, GOT and GPT in grains played important roles in nitrogen metabolism at the grain-filling stage.
Since the decrease of GS activity in grains did not influence the accumulations of amino acids and protein, it is implied that
GS might not be the key enzyme in regulating glutamine content in grains.
Key words: high temperature; nitrogen metabolism enzyme; protein; amino acid; rice
Rice (Oryza sativa L.) is a thermophilic crop mainly
produced and consumed in Asia. It is extensively
grown in the warmer, high radiation post-monsoon
and summer months. However, high temperature (HT)
will reduce rice yield and quality at the grain-filling
stage (Peng et al, 2004). Recent global warming is
likely to exacerbate rice production, with the changes
in warm extremes generally follow the changes in the
average summertime temperature, especially during
the time from late July to early August when rice is at
flowering and grain-filling (Kharin et al, 2007; Zou et al,
2009). Much research has studied the effects of HT on
rice output character, grain quality and carbohydrate
metabolism (Resurreccion et al, 1977; Jagadish et al,
2007; Teng et al, 2008; Li et al, 2010), but little is
known on nitrogen metabolism in grains (Yamaya et al,
1992; Lu et al, 2002). The mechanism in changes of
protein content, amino acid content and key enzyme
activity in nitrogen metabolism has not been very
clear up to date.
Nitrogen metabolism plays an important role in
plant growth and development. Inorganic nitrogen can
be absorbed and used only after being assimilated to
organic nitrogen, of which glutamate and glutamine are
the most important assimilation metabolites synthesized
from ammonia. Glutamine synthetase (GS)/glutamate
synthase (GOGAT) was found to catalyze the ammonia
assimilation (Lea and Mifiln, 1974) and proved to be
the main pathway in ammonia assimilation in higher
plants (Hirel et al, 2001; Miflin et al, 2002; Glevarec
et al, 2004; Martin et al, 2006). The transamination
reactions that transfer amino groups from glutamate to
other amino acids play important roles in nitrogen
metabolism (Lea et al, 1992). Glutamic oxalo-acetic
transminase (GOT), the most active aminotransferase,
catalyzes the reaction to produce aspartate which is
the precursor of aspartate family amino acids. Also,
glutamate pyruvate transminase (GPT) is an important
enzyme which catalyzes the reaction to produce
alanine.
Grain-filling is the most sensitive stage to
environmental conditions for rice development.
During this period, daily average temperature would
be the most important factor affecting rice quality (Lin,
1994; Cheng et al, 2002). Since rice nutrition quality
is valuable for the contents of protein and amino acids,
the effects of HT on nitrogen metabolism enzyme
activities and contents of protein and amino acids in
grains at the rice grain-filling stage were investigated. Received: 13 August 2010; Accepted: 24 February 2011 Corresponding author: LI Tian ([email protected])
LIANG Cheng-gang, et al. High Temperature Affects Nitrogen Metabolism Enzyme Activities and Grain Nutritional Quality 211
MATERIALS AND METHODS
Rice growth condition and treatment
Experiments were conducted at the Sichuan
Agricultural University, Ya’an City, Sichuan Province,
China in 2008. Seeds of rice varieties Koshihikari
(japonica) and IR72 (indica) were sown on 8 April
and seedlings were transplanted on 22 May. Twenty
black plastic pots (with 30 cm diameter and 30 cm
height) for each variety were used when the plants
were at the booting stage with three holes in a pot and
two seedlings in a hole. Each pot was filled with 15.0
kg cultivated soil from the same field, and applied
with 5 g (NH4)2SO4 (urea), 3 g muriate of potash (KCl)
and 6.5 g single super phosphate (SSP) as base
fertilizer without top-dressing. When the rice plants
were at the heading stage, four pots of each variety
were moved into four phytotrons (PGXZ-310D;
Ningbo East Instrument Co., Ltd, Zhejiang, China) to
expose high temperature (day/night temperature was
35 ºC/29 ºC), and the other 16 pots of each variety
were divided into four groups to grow in the field at
normal temperature (NT). The day/night temperatures
were 26.3 ºC/19.8 ºC for Koshihikari, and 25.9 ºC/
19.1 ºC for IR72, respectively.
Sampling
At the initial heading stage, plants with identical
heading date were selected and marked with a
waterproof pen to record their respective heading date
on a plastic label. The marked spikelets were sampled
once every 7 d during the period from the 7th day after
heading to ripening. Three pots of each variety with
three label-marked spikelets for each pot under both
HT and NT were collected at 9:00–9:30 a.m., and
were immediately wrapped with aluminium foil and
frozen in liquid nitrogen, and then quickly placed into
a sealed plastic bag and stored at -80 ºC. The heading
spikelets were sampled for measurement of enzyme
activities and soluble protein content with three
replications.
At harvest, the grains of each variety in the same
replication were collected together and put into a
drying oven at 80 ºC to constant weight. Then grains
were processed with a JLGJ-45 power rice huller and
refined with a JNMJ-3 rice milling machine, crushed
and sieved with a 100-mesh sieve for the measurement
of nutritional quality.
Assay of GS activity, GOGAT activity and soluble
protein content
Fifteen labeled grains were dehulled and
weighted. Added with 5 mL of buffer solution (pH 7.5,
0.1 mol/L Tris-HCl, 5 mmol/L 2-mercaptoethanol and
2 mmol/L EDTA) pre-cooled in ice, the grains were
ground into homogenate and centrifugated at 10 000
r/min for 15 min at 4 ºC. The supernatant was used for
enzyme and solution protein assays (Cai et al, 2007;
Zhao et al, 2008).
GS activity
One hundred microlitre enzyme solution was
added into 0.5 mL reaction solution (pH 7.2, 100
mmol/L imidazole-HCl, 20 mmol/L MgCl2, 25 mmol/L
2-mercaptoethanol, 50 mmol/L sodium L-glutamate,
125 mmol/L hydroxylamine and 10 mmol/L ATP).
The reaction was conducted at 35 ºC for 30 min and
terminated by adding 0.75 mL of 0.37 mol/L FeCl3,
0.2 mol/L TCA, 0.67 mol/L HCl. The OD value was
read at 535 nm 10 min later. The reaction solution
without ATP and glutamate was used as control. One
unit of GS activity is defined as the amount of enzyme
that catalyses the production of 1 μmol glutamyl-
hydroxamate per min.
GOGAT activity
One hundred microlitre enzyme solution was
added into 1 mL reaction solution (pH 7.5, 0.1 mol/L
potassium phosphate, 2 mmol/L α-oxoglutarate, 0.2
mmol/L NADH, 10 mmol/L L-glutamine). The reaction
was conducted at 30 ºC for 30 min and stopped by
boiling in water for 30 s. The OD value was read at
340 nm immediately. The reaction solution without
L-glutamine was used as control. One unit of GOGAT
activity is defined as the amount of enzyme which
consumed 1 μmol NADH per min.
Soluble protein content
Soluble protein content was measured according
to the Coomassie blue staining (Neuhoff et al, 1985).
Rice Science, Vol. 18, No. 3, 2011 212
Assay of GOT and GPT activities
Twenty labeled grains were dehulled and
weighted. Added with 5 mL of buffer (pH 7.2, 0.2
mol/L Tris-HCl) pre-cooled in ice, the grains were
ground into homogenate and centrifugated at 10 000
r/min for 20 min at 4 ºC. The supernatant was diluted
four times for the enzyme assays (Wu et al, 1998).
One hundred microlitre enzyme solution was
added into 0.5 reaction solutions, respectively (GOT
reaction solution: pH 7.4, 200 mmol/L L-aspartic acid,
2 mmol/L α-oxoglutarate, and 1 mol/L NaOH; GPT
reaction solution: pH 7.4, 200 mmol/L L-lactamine, 2
mmol/L α-oxoglutarate, 0.1 mol/L phosphate buffer,
and 1 mol/L NaOH). The reaction was conducted at 37 ºC
for 30 min and stopped by adding 0.5 mL of 1
mmol/L 2,4-dinitrophenylhydrazine. After that, 0.5
mL of reaction solution was added to react at 37 ºC
for 20 min. The reaction was stopped by adding 5 mL
of 0.4 mol/L NaOH. The OD value was read at 500
nm 10 min later. The reaction solution with GOT and
GPT solution added after 2, 4-dinitrophenylhydrazine
was used as control, respectively. One unit of GOT
(GPT) activity was defined as the amount of enzyme
that catalyses the production of 1 μmol pyruvic acid
per min.
Assay of rice nutritional quality
Protein content
Three hundred milligram sample was dissolved
in 10 mL of H2SO4 to nitrify at 380 ºC for 2 h. The
cooling solution was assayed by a kjeltec protein
analyzer (B-324, BÜCHI Labortechnik, Switzerland)
and the conversion coefficient was 5.95.
Amino acid content
Two hundred milligram sample was dissolved in
8 mL of 6 mol/L HCl at 110 ºC for 22 h. The sample
was filtered to remove insoluble materials and added
with water to 25 mL and mixed well. One millilitre of
the mixture was evaporated and the dried material was
re-dissolved in 3 mL of 0.02 mol/L HCl, 20 μL of
which were injected into an amino acid analyzer
(L-8800, Hitachi Instruments Engineering, Tokyo,
Japan) to assay amino acid content. For the acidolysis
of HCl, tryptophan could not be measured.
RESULTS Effects of high temperature on nitrogen metabolism
enzyme activities in grains at grain-filling stage
Dynamic changes of GS and GOGAT activities in grains
GS and GOGAT, the most important enzymes in
nitrogen metabolism, regulate the contents of glutamine
and glutamate. As shown in Fig. 1, GS and GOGAT
activities in grains changed in the pattern of a unimodal
curve at the grain-filling stage, which increased
gradually to peak value and thereafter descended.
Under HT, the average activity (the average of 5 times
measurements, the same below) of GS significantly
decreased in grains of Koshihikari and highly
significantly decreased in grains of IR72. However,
the average activity of GOGAT significantly increased
in grains of Koshihikari and highly significantly
increased in grains of IR72, as compared with that
Fig. 1. Changes of glutamine synthetase (GS) and glutamate synthase (GOGAT) activities in grains during grain filling stage under high temperature (HT) and normal temperature (NT) conditions.
LIANG Cheng-gang, et al. High Temperature Affects Nitrogen Metabolism Enzyme Activities and Grain Nutritional Quality 213
under NT. The glutamine catalyzed by GS in grains
could not satisfy the demand of the increasing
GOGAT activity under HT, suggesting that glutamine
might be catalyzed not only by GS in grains, but also
in other organs from which it was transferred to
grains.
Dynamic changes of GOT and GPT activities in grains
As the main aminotransferases, GOT and GPT
have been widely studied. The activities of GOT and
GPT in grains during the grain-filling were tested, and
the dynamic changes are shown in Fig. 2. Under HT,
the average activities of GOT and GPT significantly
increased in grains of Koshihikari and highly significantly
increased in grains of IR72, which indicated an
enhancement of transamination in grains under HT.
Dynamic changes of soluble protein content in grains
Soluble proteins are important components to
reflect the assimilation and metabolism ability in
plants. The soluble protein content in grains both
under HT and NT during grain-filling was determined.
A descending trend of soluble protein content during
grain filling was shown in Fig. 3. Under HT, the
average content of soluble protein in grains was
highly significantly increased, which was consistent
with the results of Zhang et al (2002).
Effects of high temperature on grain nutritional
quality at grain-filling stage
Rice nutritional quality is appraised by protein
content and amino acid content (Cheng et al, 1986;
Zhen et al, 1997). As shown in Table 1, protein and
amino acid contents were highly significantly increased
under HT. Compared with those under NT, protein
content was increased by 42.86% in Koshihikari, and
50.81% in IR72, respectively. Total amino acid
content (Trp excluded), essential amino acid content
and lysine content were increased by 49.91%, 50.72%
and 72.89% in Koshihikari, and 68.34%, 67.42% and
73.62% in IR72, respectively, which suggests that HT
improved rice nutritional quality.
DISCUSSION
Amino acids in plants are derived from ammonia
assimilation catalyzed by GS and GOGAT. GS is a
multi-functional enzyme in the center of nitrogen
metabolism, and GOGAT plays an important role in
Fig. 2. Changes of Glutamic oxalo-acetic transminase (GOT) and glutamate pyruvate transminase (GPT) activities in grains during grainfilling stage under high temperature (HT) and normal temperature (NT) conditions.
Fig. 3. Changes of soluble protein content in grains during grainfilling stage under high temperature (HT) and normal temperature (NT) conditions.
Rice Science, Vol. 18, No. 3, 2011 214
nitrogen metabolism. Research has shown that there
exists significantly positive correlation between GS
activity and protein content, and the increase of GS
activity can enhance nitrogen metabolism and promote
the synthesis of amino acid (Tang et al, 1999; Zhu et al,
2001; Miflin et al, 2002; Jin et al, 2007). However, in
this study it is interesting that the GS activity in grains
significantly decreased under HT during grain-filling,
whereas the GOGAT activity and the protein content
significantly increased. The simple correlation analysis
indicated a significantly negative correlation between
GS activity and protein content, but a significantly
positive correlation between GOGAT activity and
protein content, which was consistent with the results
of Wang et al (2005). It indicates that the synthesis of
glutamate and protein in grains was not affected by
HT, and GS in grains was not the key enzyme to
regulate the glutamine content. The probable reason
would be that most of glutamines were synthesized in
source organs and transferred to grains during grain-
filling and the GS activity in grains was inhibited by
the feedback inhibition of glutamines. The plenitudinous
glutamine ensured the synthesis of glutamate
catalyzed by GOGAT, and suggested that GOGAT
might play an important role in nitrogen metabolism
in grains during grain-filling.
It is generally accepted that there exists
significantly positive correlations between protein
content and the activities of GOT and GPT (Ebeid et al,
1981; Zhu et al, 1991). However, some research
indicated that the correlation was not significant
(Dalling et al, 1976). Our results showed that the
activities of GOT and GPT were significantly
increased under HT during grain-filling and were
significantly and positively correlated with protein
content. Transamination played an important role in
nitrogen metabolism in grains during rice grain-filling.
Since aspartate catalyzed by GOT is the precursor of
lysine which is lacked in cereal grains, the increase of
GOT activity under HT indicated that HT stress may
improve the rice nutritional quality during grain-
filling.
Although genetic engineering approaches are the
preferred techniques to increase protein and amino
acid contents (Jiao et al, 2008; Ufaz and Galili, 2008),
unfortunately, it is mostly unsuccessful to identify
opaque-2 genotypes (high-lys maize mutation) in
other crop species. The inter-regulation of metabolism
with plant stress physiology could lead to successful
nutritional improvements (Galili, 2011). In this study,
the contents of protein and amino acids in grains were
highly significantly increased under HT. Protein
content, total amino acid content (Trp excluded),
essential amino acid content and lysine content were
increased by 42.86%, 49.91%, 50.72% and 72.89% in
Koshihikari, and 50.81%, 68.34%, 67.42% and
73.62% in IR72, as compared with those under NT,
respectively. Since nitrogen metabolism supplies
glutamine, glutamate and aspartate which are the
precursors for the synthesis of other amino acids,
especially the essential amino acids, further analyses
of the accumulation of amino acids, the precursors for
the biosynthesis as well as products of the degradation
of protein, are required during rice grain filling under
HT.
ACKNOWLEDGEMENTS
This work was financed by the International
Technological Cooperation Program of Science and
Technology Department, Sichuan Province, China
Table 1. Effects of high temperature on the contents of protein and amino acids in grains at the grain-filling stage. mg/g
Koshihikari IR72 Amino acid and protein HT NT
HT NT
Nonessential amino acid 9.448 A 5.866 B 9.635 A 5.198 B6.805 A 4.571 B 6.957 A 4.275 B
19.020 A 13.281 B 20.413 A 11.980 B5.108 A 3.252 B 5.036 A 3.136 B6.970 A 4.136 B 7.020 A 3.591 B2.732 A 1.841 B 2.732 A 1.737 B3.113 A 1.902 B 3.124 A 1.773 B9.508 A 5.913 B 9.577 A 5.363 B8.160 A 6.011 B 8.063 A 5.352 B
Asp Ser Glu Gly Ala Tyr His Arg Pro Cys 2.747 A 2.470 B 3.029 A 2.340 B
Essential amino acid 3.331 A 2.355 B 3.711 A 2.225 B7.387 A 4.576 B 7.356 A 4.303 B2.361 A 1.586 B 3.338 A 1.650 B4.737 A 3.146 B 4.669 A 2.931 B9.520 A 6.587 B 9.320 A 5.852 B6.413 A 4.606 B 6.376 A 3.939 B
Thr Val Met Ile Leu Phe Lys 5.440 A 3.146 B 5.048 A 2.907 B
Total amino acid 112.799 A 75.243 B 115.403 A 68.552 BProtein 93.100 A 65.167 B 89.533 A 59.367 B
For the same variety under different treatments, values followed by the same capital letters are significant at 1% probability level.
HT, High temperature; NT, Normal temperature.
LIANG Cheng-gang, et al. High Temperature Affects Nitrogen Metabolism Enzyme Activities and Grain Nutritional Quality 215
(Grant No. 2010HH0015), and the Science and
Technological Innovation Project for Youth of Sichuan
Agriculture University, China (Grant No. 04030100).
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