全 文 ：Vol. 30 , No. 10
pp. 975 - 979 　Oct. , 2004
作 　物 　学 　报
ACTA AGRONOMICA SINICA
第 30 卷 第 10 期
2004 年 10 月 　975～979 页
Effects of Varied Carbon and Nitrogen Supply on Starch and Protein Accumulation
in Grain during Early Filling of Wheat
ZHOU Qin , J IANG Dong , DAI Ting2Bo , CAO Wei2Xing 3 , J ING Qi
( Key Laboratory of Crop Growth Regulation , Ministry of Agriculture , Nanjing Agricultural University , Nanjing 210095 , Jiangsu , China)
Abstract 　In vitro ear culture experiments were conducted to determine how the concentrations and combinations of sucrose
and glutamine in medium were suitable for the accumulation of grain starch and protein in winter wheat (cv. Ningmai 9) .
The results showed that with the same level of glutamine supply , the grain weight and starch yield were increased but the
protein formation was inhibited with increasing sucrose supply significantly. While with the same sucrose supply , both
starch and protein accumulation in grains were increased with glutamine supply increase. Higher sucrose supply led to high2
er sucrose and starch content in grains , but lower activities of Sucrose Synthase (SS) , Soluble Starch Synthase (SSS) and
Granule Bound Starch Synthase ( GBSS) , whereas higher glutamine supply led to the higher starch yield and enzyme activi2
ties. In conclusion , starch accumulation in wheat grains was controlled not only by sucrose supply , but also by nitrogen
supply. Protein accumulation in grains was positively associated with the ratio of glutamine/ sucrose in the culture media. It
suggested that controlling substrate composition during grain filling was one of the effective approaches for regulation of pro2
tein and starch formation in grains.
Key words 　Wheat ; Carbon and nitrogen supply ; Grain Growth ; Protein ; Starch ; Key enzyme
灌浆前期碳、氮供应对冬小麦籽粒蛋白质和淀粉积累的影响
周　琴　姜　东　戴廷波　曹卫星 3 　荆　奇 Ξ
(南京农业大学农业部作物生长调控重点开放实验室 ,江苏南京 210095)
摘 　要 　采用花后离体穗培养技术 ,研究蔗糖和谷氨酰胺不同浓度配比供应对冬小麦 (宁麦 9 号) 籽粒淀粉和蛋白质积
累的影响。结果表明 :在同样谷氨酰胺水平下 ,提高蔗糖供应水平能显著增加粒重 ,促进籽粒中淀粉积累 ,但降低蛋白质
合成 ;在同样的蔗糖水平下 ,提高谷氨酰胺供应水平 ,既有利于籽粒中蛋白质积累 ,也促进淀粉合成。培养基中蔗糖浓度
变化与籽粒蔗糖含量和淀粉产量消长趋势一致 ,而与籽粒中蔗糖合成酶 (SS) 、可溶性淀粉合成酶 (SSS) 和颗粒束缚淀粉
合成酶 ( GBSS)活性消长趋势相反 ;培养基中谷氨酰胺浓度与籽粒淀粉产量和淀粉合成关键酶活性的变化趋势也一致。
说明籽粒中淀粉合成既受蔗糖供应水平调节 ,也受氮供应水平调节。提高基质中谷氨酰胺/ 蔗糖比例对籽粒蛋白质含量
和产量有正向调节效应。因此 ,改变小麦籽粒灌浆过程中的营养物组成是调控籽粒淀粉和蛋白质合成的有效途径之一。
关键词 　小麦 ; 碳氮供应 ; 籽粒生长 ; 蛋白质 ; 淀粉 ; 关键酶
中图分类号 : S512
　　Starch and protein are the main components of grains
in wheat ( Triticum aestivum L1) 1 The formation of grain
starch and protein during endosperm development affect
the yield and quality of wheat1 Donovan and Lee suggest2
ed that protein synthesis in grains was independent of
starch synthesis , while starch synthesis was affected by
the supply of amino acids[1 ]1 Sucrose was as not only the
precursor of starch synthesis but also the energy source for
both starch and protein syntheisis ( Cazetta et al1
1999) [2 ]1 The starch formation rate during the linear pe2
riod of endosperm growth was affected by the sucrose level
in kernels ( Singletary and Below 1990) [3 ] 1 GlutamineΞ基金项目 : 国家自然科学基金 (30170544) 、教育部博士点基金 (2000030707)和江苏省自然科学基金 (BK2002205 , BK2001063) 。
作者简介 : 周琴 (1977 - ) , 女 , 江苏盐城人 , 博士研究生。研究方向 : 作物生理生态。3 通讯作者 (Author for correspondence) :曹卫星。E2
mail : caow @njau1edu1cn
Received(收稿日期) : 2003206220 ,Accepted(接受日期) :20032122221

was a major form of organic nitrogen translocated to the
developing seeds ( Arruda and Dasilva 1979 ) [4 ] 1 In
grains , the imported glutamine could be directly incorpo2
rated into endosperm proteins and also contributed to the
synthesis of endosperm carbohydrates (Misra and Oaks
1985) [5 ]1
The physiology of grain development was complex
and far from being clear because of the difficulty of regu2
lating C and N applications to grain (Cazetta et al11999 ,
Faleiros et al11996 and Sharma et al11995) [2 ,6 ,7 ] 1 In
vitro ear culture could solve this problem and minimize
environmental influences by allowing the individual ear to
grow under defined conditions of temperature and C and N
applications1 In recent years , in vitro culture method was
used to assess the effects of N supply on starch and pro2
tein synthesis in maize kernels ( Singletary and Below
1990 , Faleiros et al1 1996 , Singletary et al1
1990) [3 ,6 ,8 ] , and partly to illustrate the regulation mech2
anism1 Thus , the aim of this study was to explore if starch
and protein accumulation in grains could be controlled by
varied C and N concentration and ratio in the culture me2
dia1
1 　Materials and methods
111 　Plant materials and in vitro ear culture
　　Winter wheat ( Triticum aestivum L1) cv1 Ningmai 9
was grown in the field of experimental station of Nanjing
Agricultural University in 2000 - 20011 Uniform ears were
tagged at flowering1 10 days after anthesis , the tagged
ears were detached and sterilized in 70 % ethanol for 1
min and 011 % HgCl2 for 10 min , rinsed with sterile dis2
tilled water , and then transplanted into culture media1
The basic components of the culture media followed the
MS culture media (Sharma et al1 1995) [7 ] except for the
concentration of glutamine and sucrose1
Five treatments with different combinations of con2
centrations of sucrose (C , 40 g/ L and 60 g/ L) and glu2
tamine (N , 1 g/ L and 3 g/ L) in the culture media were
implemented1 The basic MS media without sucrose and
glutamine was set as control (CK) , the other C/ N treat2
ments were 40 g/ L sucrose and 1 g/ L glutamine as 40S/
1G, 60 g/ L sucrose and 1 g/ L glutamine as 60S/ 1G, 40
g/ L sucrose and 3 g/ L glutamine as 40S/ 3G, and 60 g/ L
sucrose and 3 g/ L glutamine as 60S/ 3G1 Each treatment
had three replicates , and each replicate contained two
flasks1 Five ears were cultured in a 100 mL flask contain2
ing 50 mL of liquid medium and in the growth cabinet
with day/ night temperatures of 25/ 20 ℃, 16 h photoperi2
od and a PAR of 120μmol·m - 2·s - 11
The ears were harvested after 10 days of culture1
Two ears were frozen in liquid N for at least two hours and
then stored at - 40 ℃for the determination of the activi2
ties of sucrose synthase ( SS , EC 21411113) , soluble
starch synthase (SSS , EC 21411121) and granule2bound
starch synthase ( GBSS) 1 The remaining ears were dried
for the determinations of grain weight , sucrose , starch
and protein contents in grains1
112 　Assay of sucrose , starch and N
Sucrose and starch contents were determined with the
methods described by Yu and Zhang (1999) [9 ] and by
Lin and Zhu (1999) [9 ] respectively , and total N was ana2
lyzed by the semi2micro Kjeldahl method1
113 　Determination of sucrose synthase ( SS) , soluble
starch synthase ( SSS) and granule bound starch syn2
thase ( GBSS) activities
　　Grain samples were homogenized in a mortar with a
little quartz and 510 mL 50 mmol/ L Hepes2NaOH buffer ,
pH 7151 30μL of the homogenate was placed in 118 mL
extraction media and micro2centrifuged for GBSS assay1
The remained homogenate was centrifuged for 10 min at
16 000 ×g at 4 ℃ and the supernatant was used for SS
and SSS assay1 SS activity was assayed according to
Douglus (1988) [10 ] and Jiang et al1 (2002) [11 ]1 SSS and
GBSS activities were determined according to Jiang et al1
(2002) [11 ]1 At 30 ℃, 0135 mL of reaction mixture were
added to 115 mL Eppendorf tube which contained 30μL
enzyme supernatant , 50 mmol/ L of Hepes2NaOH buffer ,
pH 715 ,1166 mmol/ L , ADPG, 15 mmol/ L of DTT and 1
mg of amylopectin1 The reaction was stopped 20 min later
in a boiling water bath for 1 min1 The ADP produced by
the SSS or GBSS was then converted to ATP by adding
100μL of 40 mmol/ L PEP , 50μL of 50 mmol/ L MgCl2 ,
1 IU pyruvate kinase and 100μL of 50 mmol/ L Hepes2
NaOH buffer then incubating at 30 ℃for 30 min1 The re2
sultant ATP was determined by adding 5 mL of luciferin2
luciferasereagent to the mixture1
679 　　　作 　　物 　　学 　　报 30 卷 　

2 　Results
211 　Effects of C/ N supply on grain weight , starch
accumulation and sucrose content
21111 　Grain weight 　　With enhanced concentrations
of glutamine and sucrose in culture media , the single
grain weights increased from 8182 to 12145 mg per grain
(Table 1) 1 The grain weight in 60S/ 1G treatment was
13131 % higher than that in 40S/ 1G treatment1 Similar
pattern was observed under the glutamine concentration of
3 g/ L , the increment was 20113 % , but the differences
were not significant between 40S/ 1G and 40S/ 3G, or
60S/ 1G and 60S/ 3G1 This suggested that with the same
glutamine level , sucrose obviously enhanced single grain
weight , while with the same sucrose level , glutamine did
not enhance grain weight significantly1 Thus , the in2
creased grain weight was mainly associated with enhanced
C supply , rather than N supply1
21112 　Starch yield 　　Compared with CK, the starch
yields ( starch weight per grain) in the other four treat2
ments were increased by 4138 , 5126 , 516 and 6182 mg
per grain (Table 1) respectively by the supply of C and
N1 The starch yields of 60S/ 1G and 60S/ 3G were 15 %
and 17 % higher than those of 40S/ 1G and 40S/ 3G re2
spectively1 Also the starch yields of 40S/ 3G and 60S/ 3G
were 11 % and 13 % higher than those of 40S/ 1G and
60S/ 1G respectively1 This indicated that both C and N
supplies were favorable to starch accumulation in wheat
grains1
Table 1 Effects of sucrose/ glutamine ( C/ N) supply on grain
weight , starch yield , starch content and
sucrose content in wheat grains
Treatment
Grain
weight
(mg/ grain)
Starch
yield
(mg/ grain)
Starch
content
( %)
Sucrose
yield
(mg/ grain)
CK 8120 c 3198 d 48148 c 1124 c
40S / 1G 17102 b 8136 c 49111 c 1159 b
40S / 3G 17119 b 9124 bc 53175 a 1178 b
60S / 1G 19129 a 9158 b 49167 bc 2104 a
60S / 3G 20165 a 10180 a 52130 ab 2105 a
　　Note : Figures with the same letter are not different at a level of signifi2
cance of P = 01051 It is the same to Table 21
21113 　Starch content 　　When the glutamine concen2
trations in the media were 1 g/ L , the starch yields with
the sucrose supply of 40 g/ L or 60 g/ L were slightly
higher than that of CK ( Table 1) 1 However , the starch
yields of 40S/ 3G and 60S/ 3G increased by 1019 % and
719 % respectively , compared with CK1 This indicated
that high concentration of glutamine favored the increment
of starch content in grains1
21114 　Sucrose content 　　At the same level of glu2
tamine supply , the sucrose content in grain was increased
significantly with increasing sucrose concentration in cul2
ture media (Table 1) 1 With the same concentration of su2
crose , the sucrose contents in grains showed no significant
differences between treatments with different glutamine
supplies1
212 　Effects of C/ N supply on activities of SS , SSS
and GBSS
　　Activities of SS , SSS and GBSS were much higher
with C and N supply in culture media than those of CK
( Fig11) , which indicated that sucrose and glutamine sup2
ply promoted starch synthesis in grains1 When glutamine
supplies were 1 g/ L or 3 g/ L , the activities of SS , SSS
and GBSS were decreased with the increment of sucrose
supply from 40g/ L to 60 g/ L1 With the same sucrose
supply of 40 g/ L , the activities of SS , SSS and GBSS
were higher in the treatment with glutamine supply of 3 g/
L than that of 1 g/ L , which indicated that there was a
stimu2lating effect on starch synthesis by glutamine1 With
sucrose supply of 60 g/ L , SSS activity was much higher ,
while SS activity was much lower and GBSS activity
slightly lower in the treatment with glutamine supply of 3
g/ L than those of 1 g/ L1 This implied that with high su2
crose supply (60 g/ L) , glutamine might exert a limited
regulation on the activities of enzymes for starch synthe2
sis1
213 　Effects of C/ N supply on protein accumulation
in grain
21311 　Protein yield 　　Protein yields (protein weight
per grain) of 40S/ 1G, 40S/ 3G, 60S/ 1G and 60S/ 3G
were 136 % , 154 % , 128 % and 144 % of CK respective2
ly1 With glutamine supply of 1 g/ L , the grain protein
yield in the treatment with 40 g/ L in the culture media
was 6167 % higher than that with 60 g/ L sucrose1 With
glutamine supply of 3 g/ L , the grain protein yield in the
treatment with sucrose supply of 40 g/ L was 6142 % high2
er than that of 60 g/ L1 This implied that sucrose supply
779　10 期 ZHOU Qin et al . : Effects of Varied Carbon and Nitrogen Supply on Starch and Protein Accumulation in Grain . . . 　　　

generally decreased protein accumulation at the same glu2
tamine supply level1 Protein yield of grain with 3 g/ L glu2
tamine supply was 1312 % higher than that with 1 g/ L
glutamine1 It showed that glutamine favored protein accu2
mulation1
Fig11 Effects of sucrose/ glutamine( C/ N) supply on the
activities of SS , GBSS and SS in wheat grains
Table 2 Effects of sucrose/ glutamine ( C/ N) supply on
protein yield and content in wheat grains
Treatment
Protein yield
(mg/ grain)
Protein content
( %)
CK 1129 d 15178 a
40S / 1G 1176 bc 10135 c
40S / 3G 1199 a 11157 b
60S / 1G 1165 c 8157 d
60S / 3G 1187 ab 9104 d
21312 　Protein content 　　CK produced the highest
protein content among the five treatments ( Table 2) 1
With the same glutamine concentration in culture media ,
the protein content was much lower in the treatment with
60 g/ L sucrose than that with 40 g/ L sucrose , while with
the same sucrose supply , the protein content was higher
in the treatment with 3 g/ L glutamine than that with 1 g/
L glutamine1 This suggested that glutamine and sucrose
had different effects on protein accumulation in wheat
grains1
3 　Discussion
The results showed that the sucrose content in wheat
grains was higher with a sucrose concentration of 60 g/ L
in culture medium than those of 40 g/ L ( Table 1) , this
was consistent with the observations by Cazetta et al1
(1999) [2 ] and Liang et al1 ( 1993) [12 ] 1 Assimilation
availability during cereal grain development had been
taken as one factor , which might strongly affect starch
production in the endosperm ( Singletary and Below
1990) [3 ]1 With more sucrose supply in culture media ,
starch accumulation in the grain was increased (Table 1) 1
Yet within the extent of sucrose concentrations from 40 g/
L to 60 g/ L in the culture media , SS activity was de2
creased by the higher sucrose concentration1 This was re2
lated to the response of SS to substrate supply1 Cazetta et
al1 (1999) [2 ] reported a similar response of soluble in2
vertase activity in maize kernel cultured in vitro to the su2
crose levels in the medium1 The low SS activity at high
media sucrose might be related to the adequate levels of
sucrose for optimal kernel growth ( Cazetta et al1
1999) [2 ]1 So the low activity of SS in this study could be
the result of negative feedback effect of the metabolic
product of sucrose1 SSS and GBSS also showed similar re2
sponses to the change in sucrose level1 With the same su2
crose level of 40 g/ L , the activities of SS , SSS and GBSS
in the treatment with glutamine supply of 3 g/ L were
higher than that of 1 g/ L (Fig11) 1 This could be related
to that N supply promoted the synthesis of key enzymes of
starch metabolism ( Singletary and Below 1989 ) [13 ] ,
which resulted in high starch yields (Table 1) 1 Since SS ,
SSS and GBSS played key roles in starch synthesis (Jiang
et al1 2002 , Li et al1 2001) [11 ,14 ] , it could be conclud2
ed that starch accumulation in wheat grain was controlled
not only by sucrose supply , but also by the activities of
key enzymes1
Composition of nutrients translocated from plant to
developing grain could influence the final composition of
assimilation stores in seeds (Reggiani et al1 1985) [15 ]1 C
and N in kernel were interrelated and required a proper
balance between the supply of sugar and amino acids
(Singletary and Below 1989) [13 ]1 In this study , only the
starch yield was consistent with grain weight in all treat2
ments , it showed that the difference in grain protein and
starch content were not simply a diluting effect1 Further
study showed that both protein content and protein yield
in grains were the highest in the treatment of 40S/ 3G,
while the lowest in 60S/ 1G1 Starch content was also the
highest in 40S/ 3G, but rated as the third in 60S/ 1G1
Obviously the condensing or dilution effects of starch
could not well explain the high protein content in grains in
40S/ 3G and the low protein content in 60S/ 1G also1
879 　　　作 　　物 　　学 　　报 30 卷 　

Since the ratio of C/ N substrate in the culture media (or
in the endosperm of grains) regulated the synthesis of
starch and protein , and subsequently determined , to a
great extent , the contents of starch and protein in grains ,
there was a strong correlation between the C/ N ratios of
the medium and the endosperm formation (Singletary and
Below 1989) [13 ] 1 This also supported our previous pro2
posal that protein content in grains was little related to the
amount of N redistribution from vegetative organs to
grains , but more negatively related to the ratio of redis2
tributed sugar/ nitrogen , while starch content positively
related to the ratio of redistributed sugar/ nitrogen ( Zhou
et al1 2002 ) [16 ] 1 Further investigations , of course ,
should be conducted to determine the proper ratio of su2
crose/ glutamine in the culture media or in the supply sub2
strate of the endosperm for regulating protein and starch
deposition in wheat grains1
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勘　　误
本刊 2004 年第 7 期 680～685 页的“陆地棉分子标记辅助轮回选择聚合育种研究 IV. 纤维比强度选择效
果及对其他品质性状的影响”英文摘要里的数据需更改。原文是 :the fiber strength means of populations with and
without RAPD ubc301 was 31. 19 cN/ tex and 29. 83 cN/ tex , as the two markers were used simultaneously , the means
was 31. 66 cN/ tex and 30. 80 cN/ tex , respectively. 应改为 the fiber strength means of populations with and without
RAPD ubc301 was 31. 55 cN/ tex and 30. 79cN/ tex , as the two markers were used simultaneously , the means was 32. 03
cN/ tex and 30. 80 cN/ tex , respectively.
979　10 期 ZHOU Qin et al . : Effects of Varied Carbon and Nitrogen Supply on Starch and Protein Accumulation in Grain . . .