Activities of Key Enzymes in Sucrose-to-Starch
Conversion in Wheat Grains Subjected
to Water Deficit during Grain Filling1
Jianchang Yang, Jianhua Zhang*, Zhiqing Wang, Guowei Xu, and Qingsen Zhu
College of Agriculture, Yangzhou University, Yangzhou, Jiangsu, China 225009 (J.Y., Z.W., G.X., Q.Z.);
and Department of Biology, Hong Kong Baptist University, Hong Kong, China (J.Z.)
This study tested the hypothesis that a controlled water deficit during grain filling of wheat (Triticum aestivum) could accelerate
grain-filling rate through regulating the key enzymes involved in Suc-to-starch pathway in the grains. Two high lodgingresistant wheat cultivars were field grown. Well-watered and water-deficit (WD) treatments were imposed from 9 DPA until
maturity. The WD promoted the reallocation of prefixed 14C from the stems to grains, shortened the grain-filling period, and
increased grain-filling rate or starch accumulation rate (SAR) in the grains. Activities of Suc synthase (SuSase), soluble starch
synthase (SSS), and starch branching enzyme (SBE) in the grains were substantially enhanced by WD and positively correlated
with the SAR. ADP Glc pyrophosphorylase activity was also enhanced in WD grains initially and correlated with SAR with
a smaller coefficient. Activities of granule-bound starch synthase and soluble and insoluble acid invertase in the grains were
less affected by WD. Abscisic acid (ABA) content in the grains was remarkably enhanced by WD and very significantly
correlated with activities of SuSase, SSS, and SBE. Application of ABA on well-watered plants showed similar results as those
by WD. Spraying with fluridone, an ABA synthesis inhibitor, had the opposite effect. The results suggest that increased grainfilling rate is mainly attributed to the enhanced sink activity by regulating key enzymes involved in Suc-to-starch conversion,
especially SuSase, SSS, and SBE, in wheat grains when subjected to a mild water deficit during grain filling, and ABA plays
a vital role in the regulation of this process.
Starch in the endosperm of wheat (Triticum aestivum)
is the major form of carbon reserves and comprises
65% to 75% of the final dry weight of the grain
(Housley et al., 1981; Dale and Housley, 1986;
Hurkman et al., 2003). Grain filling is therefore mainly
a process of starch biosynthesis and accumulation. It
is generally accepted that four enzymes may play a
key role in this process: Suc synthase (SuSase; EC
2.4.1.13), ADP Glc pyrophosphorylase (AGPase; EC
2.7.7.27), starch synthase (StSase; EC 2.4.1.21), and
starch branching enzyme (SBE; EC 2.4.1.18; Hawker
and Jenner, 1993; Ahmadi and Baker, 2001; Hurkman
et al., 2003). SuSase catalyses the cleavage of Suc, the
main transported form of assimilates in wheat plants
(Fisher and Gifford, 1986), to form UDP-Glc and Fru,
which is thought to be the first step in the Suc-to-starch
conversion. Its activity is considered to be linked to
sink strength in the developing rice (Oryza sativa) grain
and tomato (Lycopersicon esculentum) fruit (Sun et al.,
1992; Wang et al., 1993; Kato, 1995). AGPase produces
ADP-Glc, the primer of the starch chain (Smith and
1
This work was supported by the Research Grant Council of
Hong Kong (RGC 2052/00M), by the Area of Excellence for Plant
and Fungal Biotechnology in the Chinese University of Hong Kong,
and by the State Key Basic Research and Development Plan (grant
no. G1999011704).
* Corresponding author; e-mail [email protected]; fax 852–
3411–5995.
Article, publication date, and citation information can be found at
www.plantphysiol.org/cgi/doi/10.1104/pp.104.041038.
Denyer, 1992), and is regarded as the rate-limiting
enzyme in starch biosynthesis (Preiss, 1988). StSase,
composed of both soluble and granule-bound isoforms, elongates the amylose and amylopectin chains
(Déjardin et al., 1997). Soluble StSase (SSS) activity is
reported to be positively correlated with the rate of
starch synthesis in wheat grains (Keeling et al., 1993).
SBE forms branches on the polymers. It cleaves a-1,4
bonds on both amylose and amylopectin molecules
and reattaches the released glucan segments to the
same or another glucan chain through the formation
of a-1,6 linkages (Hurkman et al., 2003). Its activity
is closely associated with the increase in starch content during the development of rice endosperm
(Nakamura et al., 1989; Nakamura and Yuki, 1992).
Extensive studies have been done on the effects of
heat stress on the activities of enzymes involved in
Suc-to-starch metabolism in cereals (Caley et al., 1990;
Hawker and Jenner, 1993; Keeling et al., 1993; Jenner,
1994; Cheih and Jones, 1995; Duke and Doehlert, 1996;
Wilhelm et al., 1999; Hurkman et al., 2003). A correlation of reduction in starch content with declines in
SuSase and SSS activities in heat-treated grains has
been reported in these studies. With the exception of
the work done by Ahmadi and Baker (2001), who
reported that reduction in grain growth rate of waterstressed wheat plants resulted from a reduced SSS
activity, whereas growth cessation was due mainly to
the inactivation of AGPase, information is scarce on
changes in activities of key enzymes in Suc-to-starch
catalytic pathway in water-stressed wheat grains dur-
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Yang et al.
22.50 MPa at 31 DPA. However, the differences in
predawn (6 AM) leaf water potentials between WW and
WD plants were insignificant (Fig. 1), indicating that
plants subjected to water deficit could rehydrate overnight.
Carbon Remobilization and Grain-Filling Rate
Figure 1. Changes of leaf water potentials of wheat during the first
22 d after withholding water. The treatments are NN 1 WW (d), NN 1
WD (s), HN 1 WW (n), and HN 1 WD (h). Measurements were made
on the flag leaves at predawn (6 AM, dashed lines) and at midday (11:30
AM, solid lines). Vertical bars represent 6SE of the mean (n 5 12) where
these exceed the size of the symbol.
ing the filling period. Our early work (Yang et al., 2000,
2001b) has shown that a controlled water stress imposed during the grain-filling period of wheat and rice
can enhance remobilization of prestored carbon reserves to grains and accelerate grain filling. However,
little is known about whether and how the sink
strength is involved in these processes. The objective
of this study was to test the hypothesis that a controlled
water deficit during the grain filling may enhance sink
strength by regulating the key enzymes involved. The
changes in activities of SuSase, AGPase, StSase, SBE,
and acid invertase (AI; EC 3.2.1.26), a possible Succleaving enzyme, in wheat grains and their relationship with grain filling were investigated.
Abscisic acid (ABA) is generally regarded as a very
sensitive signal produced during water stress (Zhang
and Davies, 1990a, 1990b; Davies and Zhang, 1991).
Our earlier work has shown that an increased grainfilling rate is closely associated with an enhanced ABA
level in water-stressed grains (Yang et al., 2001a, 2001c).
Here in this experiment, the change in ABA content
in the grains and its possible role in the regulation of
the enzymatic activities were also investigated.
At early grain-filling stage (0–12 DPA), the main form
of nonstructural carbohydrate in the stem of wheat was
fructan, Suc, Glc, and Fru (56%, 25%, 10%, and 9%,
respectively), and the activities of fructan exohydrolase
and Suc phosphate synthase in the stem were substantially enhanced by WD (data not shown). The WD
facilitated the reallocation of preanthesis assimilates
from the stems to grains. Figure 2 shows the disappearance of preanthesis-assimilated 14C in the stems
and its appearance in the grains during grain filling. At
the start of water withholding (9 DPA), about 70% of
14
C fed to the flag leaves at the booting stage was
partitioned in the stems and about 5% in the grains.
After 24 d (33 DPA), 14C in the stem was reduced by
23% to 34% under WD and 50% to 61% under WW
treatments. Opposite to that observed in the stem, the
14
C in the grains increased by 39% to 50% for WD plants
RESULTS
Leaf Water Potential
Figure 1 illustrates the progression of leaf water
potentials during the first 22 d after withholding
water. When plants were well-watered (WW), midday (11:30 AM) leaf water potentials decreased gradually during grain filling, from 20.96 million Pa (MPa)
at the beginning (9 DPA) to 21.43 to 21.55 MPa on 22 d
after withholding water (31 DPA). The water-deficit
(WD) treatment substantially reduced mid-day leaf
water potentials from 20.98 MPa at 9 DPA to 22.31 to
Figure 2. Changes of 14C partitioning in the stems (A) and grains (B) of
wheat. The 14C was fed to the flag leaves at the booting stage. The
treatments are NN 1 WW (d), NN 1 WD (s), HN 1 WW (n), and
HN 1 WD (h). Arrows in the figure indicate the start of withholding
water. Vertical bars represent 6SE of the mean (n 5 12) where these
exceed the size of the symbol.
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Water Deficit and Enzyme Activity in Wheat Grains
similar observation was made on grain-filling rate
(Table I). The WD substantially increased the grainfilling rate and shortened the grain-filling period at
both NN and NH. The active grain-filling period was
shortened by 8 d at NN and 10 d at HN, and grainfilling rate increased by 0.54 mg per day per kernel at
NN and 0.74 mg per day per kernel at HN, respectively, compared with their respective WW treatments.
The final grain weight was not significantly different
between the WW and WD treatments when NN was
applied (Table I). However, it was significantly increased under HN plus WD treatments. A similar
result was obtained with grain yield because only the
kernel weight, rather than the spike number per m2 or
grain number per spike, was influenced by the WD
during grain filling in this experiment (Table I).
Changes in Activities of Enzymes
Figure 3. Starch accumulation process (A) and SAR (B) of wheat grains.
The treatments are NN 1 WW (d), NN 1 WD (s), HN 1 WW (n), and
HN 1 WD (h). SAR was calculated according to Richards’ (1959)
equation. Arrows in the figure indicate the start of withholding water.
Vertical bars in A represent 6SE of the mean (n 5 6) where these exceed
the size of the symbol.
and only 9% to 18% for WW ones at 33 DPA. In
comparison, application of a high amount of N (HN)
reduced the 14C reallocation into the grains (Fig. 2).
The WD greatly accelerated starch accumulation in
grains from 9 to 27 DPA at normal amount of N (NN)
and from 12 to 33 DPA at HN (Fig. 3). Eighty-one
percent to 92% of the final starch weight in WD grains
was accumulated during this period, and only 44% to
52% for WW grains in the same period. HN slowed the
starch accumulation either for WW or WD plants. A
The activities of the five enzymes examined in
relation to Suc-to-starch conversion in wheat grains
exhibited variable responses with time, soil moisture,
and N levels. SuSase activity (assayed in cleavage
direction) in WW grains was increased from 9 to 27
DPA and then decreased with grain development
(Fig. 4A). It was substantially enhanced by WD during
the first 9 to12 d after withholding water, reached its
peak 18 and 21 DPA at NN and HN, respectively, and
sharply declined thereafter, in good agreement with
the starch accumulation rate (SAR; refer to Fig. 3B).
Irrespective of NN and HN treatments, activities of
both soluble and insoluble AI in WW grains were little
changed from 9 to 18 DPA and decreased afterward
(Fig. 4, B and C). They were enhanced by WD, with
soluble AI enhanced more than insoluble AI. SuSase
activity, on a per grain basis, was much higher than
those of AI during the rapid accumulation period of
starch in the grain (9–27 DPA at NN and 12–33 DPA at
HN, respectively, for WD plants and 12–39 DPA for
WW plants; refer to Fig. 3), indicating that SuSase is
a predominant enzyme responsible for Suc cleavage in
wheat grains.
Very similar to SuSase activities, SSS and SBE
activities were markedly increased in WD grains
Table I. Grain-filling rate and grain yield of wheat subjected to various N and soil moisture treatments
The treatments are NN 1 WW, well watered with normal amount of nitrogen; NN 1 WD, water deficit with normal amount of nitrogen; HN 1 WW,
well watered with high amount of nitrogen; and HN 1WD, water deficit with high amount of nitrogen. Active grain-filling period and grain-filling rate
were calculated according to Richards’ (1959) equation. Values of spike number per square meter, grain number per spike, and grain weight were
means of plants harvested from 2 m2 of each treatment. The grain yield was the means of plants harvested from 18 m2 of each treatment. Statistical
comparison was within the same column.
Treatment
NN
NN
HN
HN
a
1
1
1
1
WW
WD
WW
WD
Active Grain-Filling Period
Grain-Filling Rate
Spike Number
Grain Number
Kernel Weight
Grain Yield
d
mg grain21 d 21
spike m22
grain spike21
mg grain21
g m22
27
19
34
24
a
b
d
a
c
1.40 c
1.94 a
0.95 d
1.69 b
419
417
424
421
a
a
a
a
41
40
42
43
a
a
a
a
42
41
36
45
b
b
c
a
701 b
679 b
628 c
785 a
Different letters indicate statistical significance at the P 5 0.05 level of probability.
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Yang et al.
slowed down their declines at the late filling stage
when csoil was the same.
Changes in ABA Content
As shown in Figure 6, ABA content in the grains was
very low at early grain-filling stage and remarkably
enhanced by WD at both NN and HN. At early and
mid-grain-filling stages, the grain with NN contained
more ABA than those with HN treatments when soil
water potential was the same. The change in ABA
Figure 4. Changes in activities of SuSase (A), soluble AI (B), and
insoluble AI (C) in wheat grains. The treatments are NN 1 WW (d),
NN 1 WD (s), HN 1 WW (n), and HN 1 WD (h). Arrows in the figure
indicate the start of withholding water. Vertical bars represent 6SE of the
mean (n 5 6) where these exceed the size of the symbol.
during the first 9 to 12 d after withholding water (Fig.
5, B and D). The changes in activities of both SSS and
SBE were consistent with SAR (refer to Fig. 3). By
contrast, granule-bound starch synthase (GBSS) activity was little affected by either the WD or N treatments
(Fig. 5C).
AGPase activity exhibited a peak at 15 to18 DPA in
WD grains and 21 to 27 DPA in WW grains (Fig. 5A).
The peak appeared just before or corresponded to the
maximum SAR. The activity in WD grains was greater
initially and declined faster after reaching a maximum
when compared with that in WW grains. With the
exception of GBSS activity, HN reduced activities of all
the enzymes at early and mid-grain-filling stages but
Figure 5. Changes in activities of AGPase (A), SSS (B), GBSS (C), and
SBE (D) in wheat grains. The treatments are NN 1 WW (d), NN 1 WD
(s), HN 1 WW (n), and HN 1 WD (h). Arrows in the figure indicate
the start of withholding water. Vertical bars represent 6SE of the mean
(n 5 6) where these exceed the size of the symbol.
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Water Deficit and Enzyme Activity in Wheat Grains
DISCUSSION
Figure 6. Changes in ABA content in wheat grains. The treatments are
NN 1 WW (d), NN 1 WD (s), HN 1 WW (n), and HN 1 WD (h).
Arrow in the figure indicates the start of withholding water. Vertical bars
represent 6SE of the mean (n 5 6) where these exceed the size of the
symbol.
content in the grains was paralleled with SAR (refer to
Fig. 3), and they were very significantly correlated (r 5
0.99**, P 5 0.01).
Correlations of Enzyme Activities with ABA Content
and Starch Accumulation Rate
During the rapid accumulation period of starch in
the grain, the correlations of activities of the five
enzymes examined with ABA content and SAR were
analyzed (Table II). Activities of SuSase, SSS, and SBE
were positively and very significantly correlated with
SAR (r 5 0.92** to 0.97 **, P 5 0.01). AGPase activity
was also correlated with SAR with r 5 0.69* (P 5 0.05),
whereas neither of the soluble or insoluble AI nor
GBSS was significantly correlated (r 5 0.35–0.48, P .
0.05). Very similar correlations were observed between
the activities of enzymes and ABA content in the
grains (Table II), suggesting that ABA may play an
important regulative role in enzyme activities.
Effects of Exogenous ABA and Fluridone
When ABA was applied to HN plus WW plants at
early grain-filling stage (9–13 DPA), the activities of
SuSase, SSS, and SBE and starch accumulation in the
grains were very significantly increased (Table III).
Effects of ABA application on AGPase and soluble AI
activities varied with the determination date. Supplemental ABA had no significant effects on the activities
of insoluble AI and GBSS. Opposite to ABA, spraying
with fluridone, an inhibitor of ABA synthesis, significantly reduced activities of SuSase, SSS, SBE, and
AGPase and starch content in the grains (Table III). The
final grain weight was 46.1 g and 26.6 g, respectively,
for ABA- and fluridone-treated plants, which was 129%
and 75% of that (35.6 g) for the control, respectively.
Water stress imposed during the grain-filling period
of wheat, especially at the early filling stage, usually
results in a reduction in grain weight and leads to reduced grain yield (Aggarwal and Sinha, 1984; Nicolas
et al., 1985b; Kobata et al., 1992; Zhang et al., 1998). We
found, however, that if WD during grain filling was
controlled properly, plants were able to rehydrate
overnight (Fig. 1). A benefit from such a WD was that
it could enhance remobilization of carbon reserves
from vegetative tissues to the grains (Fig. 2) and accelerate SAR and the grain-filling rate (Fig. 3; Table I). The
grain weight and grain yield would not necessarily be
reduced when NN was applied (Table I). Moreover,
the grain weight and grain yield could be increased
when excessive N fertilizer was applied (Table I).
The discrepancies between our results and previous
reports (Aggarwal and Sinha, 1984; Nicolas et al.,
1985b; Kobata et al., 1992; Zhang et al., 1998) are
probably attributable to several reasons. First, the
plant materials we used in this experiment are high
yielding and strong lodging-resistant wheat cultivars,
which have shown slow grain filling and low harvest
index because they stay green for too long and
remobilize prestored assimilates poorly to the grains
(Peng et al., 1992; Yang et al., 2000). A mild WD during
grain filling would be beneficial to the grain filling by
enhancing remobilization of stored reserves from the
stems to grains for these cultivars. Second, the reduction in gain weight in response to water stress
during grain-filling period is mainly attributed to the
lowered number of endosperm cells, thereby decreasing sink size per kernel (Singh and Jenner, 1982;
Nicolas et al., 1985a; Michihiro et al., 1994). In our
experiment, WD was initiated at 9 DPA, at which time
the active division period of endosperm cells is almost
complete (Feng et al., 2000; Wang et al., 2003), and
therefore the sink size may not be seriously affected.
Third, the WD imposed in our experiments was rather
mild and controlled properly such that plants could
rehydrate overnight. We have observed that such
Table II. Relationships of ABA content and (SAR) in wheat grains with
the activities of the enzymes involved in Suc-to-starch metabolism
during the rapid SAR period (9–27 DPA for NN 1 WD, 12–33 DPA
for HN 1 WD, and 12–39 DPA for NN 1 WW and HN 1 WW
treatments)
Data used for the calculation are from Figures 3 to 6.
Correlation With
SuSase
Soluble AI
Insoluble AI
AGPase
SSS
GBSS
SBE
ABA Content
a
0.94**
0.45
0.36
0.73**
0.95**
0.47
0.98**
SAR
0.93**
0.46
0.35
0.69*
0.92**
0.48
0.97**
a
The * and ** indicate correlation significance at the P 5 0.05 and
P 5 0.01 levels of probability, respectively.
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Yang et al.
Table III. Effects of applied ABA and fluridone on ABA content (pmol grain21), activities of the enzymes involved in Suc-to-starch metabolism
(mmol grain21 h21), and starch accumulation (mg grain21) in the grains of wheat
The plants were tank grown with a HN 1 WW treatment. The spikes were sprayed either with 20 3 1026 M ABA (T1) or with 20 3 1026 M fluridone
(T2) daily for 5 d starting at 9 DPA. Control plants (CK) were sprayed with deionized water. Statistical comparison was within the same row and the
same determination date.
ABA Content/Enzyme Activities/
Starch Accumulation
ABA
SuSase
Soluble AI
Insoluble AI
AGPase
SSS
GBSS
SBE
Starch
18 DPA
CK
4.86
2.32
0.84
0.78
1.11
0.10
0.08
319
6.57
T1
a
8.93**
4.78**
1.15**
0.89b
1.78**
0.21**
0.09b
647**
9.15**
21 DPA
27 DPA
T2
CK
T1
T2
CK
T1
T2
2.37**
0.89**
0.65*
0.70b
0.86**
0.06**
0.04*
208**
4.13**
6.42
2.68
0.75
0.62
1.56
0.14
0.11
446
8.59
12.4**
6.39**
1.11**
0.74b
1.58b
0.32**
0.14b
751**
14.1**
4.76**
1.32**
0.56*
0.53b
1.27**
0.07**
0.06**
314**
6.72**
8.73
3.01
0.69
0.31
1.44
0.15
0.14
552
13.4
11.5**
4.19*
0.77b
0.23b
1.35b
0.25**
0.16b
636*
27.2**
4.21**
0.82**
0.41*
0.24b
1.23*
0.05**
0.11b
270**
9.35**
a
The * and ** indicate significantly different at the P 5 0.05 and 0.01 levels of probability, respectively.
a mild WD imposed during grain filling would not
seriously reduce photosynthesis (Yang et al., 2000,
2001a). Under such conditions, the gain from the
accelerated grain-filling rate may outweigh the possible loss of photosynthesis as a result of a shortened
grain-filling period, leading to an increase in grain
yield in the cases where plant senescence is unfavorably delayed by excessive application of N fertilizer.
It is generally accepted that grain-filling rate
in cereals is mainly determined by sink strength
(Venkateswarlu and Visperas, 1987; Liang et al., 2001).
During grain-filling period of wheat, kernels are very
strong sinks for carbohydrate (Ho, 1988; Riffkin et al.,
1995). The sink strength can be described as the
product of sink size and sink activity (Venkateswarlu
and Visperas, 1987). Sink size is a physical restraint
that includes cell number and cell size. Sink activity is
a physiological restraint that includes multiple factors
and key enzymes involved in carbohydrate utilization
and storage (Wang et al., 1993). As the sink size
was not seriously affected by the WD in this experiment, we speculate that increased carbon remobilization from the stems to grains and accelerated SAR or
grain-filling rate may be mainly attributed to an
enhanced sink activity under the WD.
It was hypothesized that high levels of enzymes
involved in the breakdown of Suc in the sink would
increase sink capacity by lowering the local concentration of Suc, thereby generating a gradient that
allows further unloading of Suc from phloem (Wardlaw, 1968; Liang et al., 2001). Since both SuSase and
invertase are involved in Suc cleavage in sink tissue,
their activities are regarded as biochemical markers of
sink strength (Wang et al., 1993; Ranwala and Miller,
1998). Our results showed that the activity of SuSase
in wheat grains was much higher than those of both
soluble and insoluble AI, and the former was enhanced more than the latter by WD (Fig. 4). SuSase
activity was positively and very significantly correlated with SAR during the rapid accumulation
period, whereas neither soluble nor insoluble AI
b
Not significantly different.
activities was positively correlated (Table II). The
results back the notion that SuSase is predominant
in the sink accumulating carbohydrate reserves and
catalyzes the first step in the conversion of Suc to
starch in the endosperm of cereals (Chourey and
Nelson, 1976; Dale and Housley, 1986; Kato, 1995),
whereas invertase is mainly present in tissues in
which active cell elongation is occurring (Sung et al.,
1988; Ranwala and Miller, 1998). The results suggest
that the enhanced carbon remobilization and grainfilling rate by a WD imposed during grain-filling
period is attributed to, or associated with, an increase
in sink strength by regulating SuSase activity in
wheat grains.
WD enhanced activities of AGPase, SSS, and SBE at
initial or at early and mid-grain-filling stages (Fig. 5).
The activities of the three enzymes were positively
correlated with SAR (Table II), suggesting that all
of the three enzymes in wheat grains play an important role in starch synthesis. We observed that GBSS
activity was neither enhanced by the WD nor significantly correlated with SAR (Table II). A probable
explanation is that SSS is thought to be responsible for
generating polymers that are the substrates for amylopectin synthesis via SBE (Smith and Denyer, 1992).
By contrast, GBSS may be involved only in amylose
synthesis. Since starch in wheat grains is composed
mostly of amylopectin, SSS is therefore considered to
play a more important role than GBSS.
It is worth noting that SBE was highly correlated
with SAR with the greatest coefficient (r 5 0.97) among
all the enzymes examined in this experiment (Table II).
The result may probably be explained by the fact that
SBE is the sole enzyme capable of forming a-1,6-linked
branches on already synthesized and/or elongating
amylose molecules (Preiss et al., 1991) and thereby
plays a key role in starch production in wheat
endosperm. A similar observation was made in rice
grains (Nakamura et al., 1989; Nakamura and Yuki,
1992). The close correlation of SBE activity with SAR in
this study suggests that accelerated grain-filling rate
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Water Deficit and Enzyme Activity in Wheat Grains
under WD is mainly associated with the enhanced SBE
activity in wheat grains.
The results that a mild WD imposed during grainfilling period could enhance activities of key enzymes
in Suc-to-starch pathway were also observed on other
wheat cultivars and rice (Yang et al., 2001a; Wang,
2003). However, the regulatory mechanisms involved
in the enzyme activities are unrevealed. We observed
from this experiment that changes in activities of
SuSase, SSS, SBE, and AGPase were closely associated
with that of ABA content (Figs. 4–6). A positive and
very significant correlation between ABA content and
activities of the enzymes was found (Table II). Furthermore, the activities of the enzymes were significantly increased when supplemental ABA was applied
to the HN plus WW plants, whereas application of
fluridone, an inhibitor of ABA synthesis, had the
opposite effects (Table III). The results suggest that
an elevated ABA level may play an important role in
the enhancement of enzymatic activities under the
WD. Little is known about how ABA activates the
enzymes. It is reported that ABA improves the energization of phloem cells in sink organs by stimulating
ATPase activity and thereby regulates the metabolism
of assimilates in cells (Peng et al., 2003). A multiplicity
of evidence shows that stress-induced ABA plays a
role in regulation of gene expression (e.g. Rock and
Quatrano, 1995). It is possible that ABA-induced gene
expression under WD may contribute to the regulative
role over the activities of enzymes involved in Suc-tostarch conversion. Further investigation is needed to
elucidate the mechanism at which ABA regulates
activities of the enzymes.
CONCLUSIONS
Our results demonstrate that if a WD stress during
the grain filling of wheat is controlled properly so that
plants can rehydrate overnight, remobilization of prestored carbon from vegetative tissues to the grains can
be substantially enhanced and SAR or grain-filling rate
be accelerated. The increased remobilization and grainfilling rate are mainly attributed to, or at least linked to,
the enhanced sink activity by regulating key enzymes
involved in Suc-to-starch pathway, especially SuSase,
SSS, and SBE, in the grains when subjected to the WD.
ABA plays a vital role in the regulation of this process.
70.4 mg kg21, respectively. On the day of sowing, 14 g N m22 as urea and
4 g phosphorus m22 as single superphosphate were applied to the soil. On
30 d after sowing (DAS) and 112 DAS, 6 g and 5 g N m22 as urea were top
dressed, respectively. The soil water content was maintained close to field
capacity (soil water potential, csoil, at 20.02 to 20.025 MPa) by manual
watering until 9 DPA when WD treatments were initiated. Yangmai 158 and
Yangmai 11 headed (50% plants) 157 DAS and 158 DAS, respectively, and
flowered during 164 to 170 DAS. Air temperatures during grain filling (164–
221 DAS), averaged 10 d, were 15.4°C, 16.9°C, 18.1°C, 19.2°C, 21.3°C, and
23.9°C, respectively.
Nitrogen and Water-Deficit Treatments
The experiment was a 2 3 2 3 2 (two cultivars, two levels of N, and two
levels of soil moisture) factorial design with eight treatments. Each of the treatments had three plots as repetitions in a complete randomized block design.
Plot dimension was 3 m 3 4 m, and plots were separated by a ridge (20 cm in
width) wrapped with plastic film. Two levels of N treatments were applied
at heading (50% of plants headed). One-half of plots were top dresses with
either 3 g N m22 (NN) or 8 g N m22 (HN) as urea. From 9 DPA (173 DAS for
both cultivars) until maturity, two levels of csoil were imposed on the plants of
both NN and HN treatments by controlling water application. The WW
treatment was maintained at 20.02MPa, and the WD treatment was maintained at 20.08 MPa. Soil water potential was monitored in the 15- to 20-cm soil
depth. Four tension meters (Soil Science Research Institute, China Academy of
Sciences, Nanjing, China) were installed in each plot to monitor. Tension meter
readings were recorded at 12 PM each day. When the reading dropped to the
designed value, 0.08 and 0.02 m3 of tap water per plot was added to the WW
and WD plants, respectively. A rain shelter, consisting of a steel frame covered
with plastic sheet, was used to protect the plot during rains.
Radioactive Labeling
At the boot stage (144 DAS for Yangmai 158 and 146 DAS for Yangmai 11),
60 plants from each treatment were labeled with 14CO2. Flag leaves of main
stems were used for the labeling between 9 AM to 11 AM on a clear day with
photosynthetically active radiation at the top of the canopy ranging between
1,000 and 1,100 mmol m22 s21. The whole flag leaf was placed into a polyethylene chamber (25-cm length and 4-cm diameter) and sealed with tape and
plasticine to maintain a gas-tight seal. Ten milliliters of air in the chamber was
drawn out, and the same volume of gas was injected into the chamber, which
contained 0.015 mol L21 CO2 at a specific radioactivity of 14C of 2.21 MBq L21.
The chamber was removed after 1 h.
Labeled plants were harvested at 0 DPA (50% anthesis) and from 9 (the
initiation of water withholding) to 54 DPA at 6-d intervals, respectively. Each
plant was divided into leaf blades, culms plus sheaths, kernels, and other
parts on a spike (rachis 1 palea 1 lemma 1 glume). Samples were dried at
80°C to constant weight, ground into powder, and then extracted by shaking
for 30 min in 80% (v/v) boiling ethanol. The residue was extracted in 2:1 of
60% (v/v) HClO4 to 30% (v/v) H2O2 for 4 h at 60°C. The radioactivity of 14C in
both the extracted aliquots was counted using a liquid scintillation counter
(Beckman Instruments, Fullerton, CA), and the data were presented in
composite averages. Radioactivity distribution in each part of the plant was
expressed as a percentage of total radioactivity remaining in the aboveground
portion of the plant.
Sampling
MATERIALS AND METHODS
Plant Materials and Cultivation
The experiment was conducted at Yangzhou University farm, Yangzhou,
China (32°30#N, 119°25#E) from November 2002 to June 2003. Two highly
lodging-resistant cultivars of semi-winter wheat (Triticum asetivum), cv
Yangmai 158 and cv Yangmai 11, currently used in local production, were
grown in the field. The sowing date was November 2, and the plant density
was adjusted to 150 plants m22 at three-leaf age. The soil of the field was sandy
loam soil (Typic fluvaquents, Entisols; U.S. taxonomy) that contained organic
matter at 2.42% and available N-phosphorus-potassium at 110, 36.5, and
A total of 150 spikes that headed on the same day were chosen and tagged
for each plot. Ten tagged spikes from each plot were sampled at 3-d intervals
from anthesis to 21 DPA and 6-d intervals from 27 DPA to maturity. All grains
from each spikelet were removed. Half of the sampled grains was frozen in
liquid nitrogen for 2 min and then stored at 280°C for enzymatic and ABA
measurements. The other half of the grains was dried at 70°C to constant
weight and weighed. The starch content in the grains was analyzed by the
method of Yoshida et al. (1976). The processes of grain filling and starch
accumulation in the grain were fitted by Richards’ (1959) growth equation as
described by Zhu et al. (1988):
W 5 A=ð1 1 Be2kt Þ
Plant Physiol. Vol. 135, 2004
1=N
ð1Þ
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Yang et al.
Both grain-filling rate and SAR (G) were calculated as the derivative of
Equation 1:
G 5 AKBe2k =ð1 1 Be2kt Þ
ðN11Þ=N
ð2Þ
where W is the grain/starch weight (mg), A is the final grain/starch weight
(mg), t is the time after anthesis (d), and B, k, and N are coefficients determined
by regression. The active grain-filling period was defined as the days when W
was from 5% (t1) to 95% (t2) of A. An average grain filling rate during this
period was therefore calculated from t1 to t2.
Plants (except border) from a 4-m2 site from each plot were harvested at
maturity for the determination of grain yield. Yield components, i.e. the spikes
per square meter, grain number per spike, and grain weight, were determined
from plants harvested from a 1-m2 site (excluding the border plants) randomly
sampled from each plot.
Measurement of Leaf Water Potential
Leaf water potentials of flag leaves were measured on clear days at
predawn (6 AM) and midday (11:30 AM) on 0, 3, 6, 10, 14, 18, and 22 d after
withholding water. Well-illuminated flag leaves were chosen randomly for
such measurements. A pressure chamber (model 3000; Soil Moisture Equipment, Santa Barbara, CA) was used for leaf water potential measurement with
six leaves for each treatment.
Enzyme Extraction and Assays
All chemicals and enzymes used for enzymatic measurement were from
Sigma (St. Louis). All enzyme assays were optimized for pH and substrate
concentration and were within the linear phase with respect to incubation
time and protein concentration. Protein content was determined according to
Bradford (1976), using bovine serum albumin as standard. Enzyme activities
were expressed on a per grain basis.
The method for SuSase and AI extraction was modified from Ranwala and
Miller (1998). For AI extraction, 30 to 40 grains were homogenized in 4 to 8 mL
of chilled 100 mM Tris buffer (pH 7.2) containing 5 mM b-mercaptoethanaol,
10 mM isoascorbate, and 1 mM phenylmethyl-sulfonyl fluoride. The homogenate was centrifuged at 15,000g for 30 min, the supernatant was dialyzed in
15 mM Tris (pH 7.2) containing 5 mM b-mercaptoethanaol overnight, and the
dialyzate was used for AI assay.
For SuSase extraction, grains were homogenized with a mortar and pestle
in 100 mM HEPES (pH 7.5) containing 10 mM isoascorbate, 3 mM MgCl2, 5 mL
dithiothreitol (DTT), 2 mL EDTA, 5% (v/v) glycerol, 3%(w/v) polyvinylpyrrolidone, and 0.01% Triton X-100. After centrifugation at 15,000g for 30 min,
the supernatant was desalted on a Sephadex G-25 column, and the proteins
were eluted by the reaction buffer that contained 50 mM HEPES (pH 7.5),
10 mM MgCl2, 2 mM EDTA, and 3 mM DTT.
The extraction procedure for AGPase, StSase, and SBE was according to
Nakamura et al. (1989). Briefly, 30 to 40 grains were homogenized with a pestle
in a precooled mortar that contained 4 to 8 mL frozen extraction medium:
100 mM HEPES-NaOH (pH 7.6), 8 mM MgCl2, 5 mM DTT, 2 mM EDTA, 12.5%
(v/v) glycerol, and 5% (w/v) insoluble polyvinylpyrrolidone 40. After being
filtered through four layers of cheesecloth, the homogenate was centrifuged
at 12,000g for 10 min, and the supernatant was used for the enzyme assay.
The enzyme activities were determined as described previously: SuSase (in
cleavage direction; Ranwala and Miller, 1998), soluble and insoluble AI
(Zinselmeier et al., 1995), SSS and GBSS (Schaffer and Petreikov, 1997), and
AGPase and SBE (Nakamura et al., 1989).
ABA Extraction, Purification, and Quantification
The methods for extraction and purification of ABA [(6)-ABA] were
modified from those described by Bollmark et al. (1988) and He (1993).
Samples of 20 to 30 grains were ground in a mortar (at 0°C) in 10 mL of 80%
(v/v) methanol extraction medium containing 1 mM butylated hydroxytoluene as an antioxidant. The extract was incubated at 4°C for 4 h and
centrifuged at 4,800g for 15 min at the same temperature. The supernatants
were passed through Chromosep C18 columns (C18 Sep-Park cartridge;
Waters, Millford, MA) and prewashed with 10 mL of 100% and 5 mL of 80%
methanol, respectively. Impurity in the extraction was checked through
dilution test and addition test as described by He (1993). The hormone
fractions were dried under N2 and dissolved in 2 mL of phosphate-buffered
saline (PBS) containing 0.1% (v/v) Tween 20 and 0.1% (w/v) gelatin (pH 7.5)
for analysis by an ELISA.
The mouse monoclonal antigen and antibody against ABA, and IgG-horse
radish peroxidase (IgG-HRP) used in ELISA were produced at the Phytohormones Research Institute, China Agricultural University, China (see He, 1993).
The method for quantification of ABA by ELISA was described previously
(Yang et al., 2001a). The recovery percentage of ABA in grains was 81.6 6 4.6.
The specificity of the monoclonal antibody and the other possible nonspecific
immunoreactive interference were checked previously and proved reliable
(Wu et al., 1988; Zhang et al., 1991; Yang et al., 2001a, 2001c).
Exogenous ABA and Fluridone Applications
Plants were grown in eight cement tanks in open field conditions. Each
tank (0.3-cm height, 1.6-m width, and 8.8-m length) was filled with sandy
loam soil with the same nutrient contents as the field soil. The sowing date and
cultivation were the same as the field experiment. A HN plus WW treatment
was conducted as described above.
Starting 9 DPA, either 20 3 1026 M (6)-ABA (Sigma) or 20 3 1026 M
fluridone (Fluka, NJ; Riedel-de Haën, Seelze, Germany), an inhibitor of ABA
synthesis, were sprayed at the rate of 800 mL m22 on the top of plants (spikes)
for 5 d. Both ABA and fluridone were applied between 4 PM and 5 PM on each
day. All the solutions contained ethanol and Tween 20 at final concentrations
of 0.1% (v/v) and 0.01 (v/v), respectively. Control plants were sprayed with
the same volume of deionized water containing same concentrations of
ethanol and Tween 20. Each treatment was an area of 2.4 m2 with four
replications.
ABA content, enzymatic activities, and starch content in the grains were
determined 3, 6, and 9 d after the chemical treatments (15, 18, and 27 DPA).
Measurement methods were the same as described above. Fifty plants from
each treatment were harvested at maturity for the determination of final grain
weight.
Statistical Analysis
The results were analyzed for variance using SAS statistical analysis
package (version 6.12; SAS Institute, Cary, NC). Data from each sampling date
were analyzed separately. Means were tested by LSD at P0.05 level (LSD0.05).
Linear regression was used to evaluate the relationships of enzymatic
activities with ABA content and SAR in the grains. Since the two cultivars
behaved the same, the data are presented as an average between the two
cultivars.
This experiment was also conducted under pot-grown conditions at the
same time as the field-grown, and results from the pot experiment were very
similar. Only the field experiment was reported in this paper because of
limited space.
Received February 14, 2004; returned for revision April 12, 2004; accepted
April 12, 2004.
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