high temperature at grain-filling stage affects nitrogen ... · from ammonia. glutamine synthetase...

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


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).


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.


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.


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.


(Grant No. 2010HH0015), and the Science and

Technological Innovation Project for Youth of Sichuan

Agriculture University, China (Grant No. 04030100).

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high temperature at grain-filling stage affects nitrogen ... · from ammonia. glutamine synthetase...

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