283

ARTICLE
Application of abscisic acid regulates antioxidant
enzymes activities and modulates endosperm cell
division in winter wheat
Dongqing Yang, Dian Peng, Wei Yang, Yanping Yin, Yong Li, and Zhen Wang
Can. J. Plant Sci. Downloaded from www.nrcresearchpress.com by 180.251.158.61 on 03/23/18

Abstract: Effects of exogenous abscisic acid (ABA) on antioxidant enzymes activities and endosperm cell division
of two wheat (Triticum aestivum L.) cultivars were investigated. Results showed that the superoxide dismutase
(SOD), peroxidase (POD), and catalase (CAT) activity in flag leaves of both cultivars is elevated by application of
ABA and it is correlated with less membrane damage: lower malondialdehyde (MDA). Exogenous ABA significantly
increased endosperm cell number and endosperm division rate, finally increased grain weight. Although ABA
treatment decreased endogenous zeatin riboside (ZR) content in flag leaves from 7 to 28 days after anthesis
(DAA), indole acetic acid (IAA) levels were significantly increased by spraying with ABA. Correlation analysis
showed that endogenous contents of ZR, ABA, and IAA in grains were positively and significantly correlated with
grain-filling rate. IAA content in leaves was positively and significantly correlated with grain-filling rate. The
results suggested that increased grain weight of ABA-treated plants was due to higher antioxidant abilities of flag
leaf resulting in longer maintenance of photosynthetic capacity and higher grain-filling rate.
For personal use only.

Key words: hormones, antioxidant enzymes, malondialdehyde, grain filling, staygreen, Triticum aestivum L.
Résumé : Les auteurs ont examiné les effets de l’acide abscissique (AA) exogène sur l’activité des enzymes antiox-
ydants et la division des cellules dans l’endosperme de deux cultivars de blé (Triticum aestivum L.). Les résultats indi-
quent que l’application d’AA augmente l’activité de la superoxyde dismutase (SOD), de la peroxydase (POD) et de la
catalase (CAT) dans les feuilles paniculaires des deux variétés, et que cette hausse est corrélée à des dommages
moins importants à la membrane cellulaire, à savoir une concentration plus faible de malondialdéhyde (MDA).
L’AA exogène accroît le nombre et la multiplication des cellules dans l’endosperme de façon significative, ce qui
a pour conséquence de donner un grain plus lourd. Bien que l’application d’AA diminue la concentration de
zéatine riboside (ZR) endogène dans les feuilles paniculaires sept à 28 jours après l’anthèse, la pulvérisation d’AA
augmente significativement la celle d’acide indole acétique (IAA). L’analyse des corrélations révèle que la teneur
endogène en ZR, AA, et IAA du grain est positivement et significativement corrélée au remplissage du grain. La
concentration d’IAA dans les feuilles présente une corrélation positive significative avec ce facteur. Les résultats
donnent à penser que le poids plus élevé du grain des plants traités à l’AA est attribuable au meilleur pouvoir anti-
oxydant de la feuille paniculaire, qui permet à la photosynthèse de se poursuivre plus longtemps et au grain de se
remplir davantage. [Traduit par la Rédaction]
Mots-clés : hormones, enzymes antioxydants, malondialdéhyde, remplissage du grain, stay-green, Triticum aestivum L.

Introduction been reported that maintenance of photosynthetic

Agricultural products demand is estimated to increase
by about 50% by 2030 due to the rapidly growing world
population and the frequent occurrence of natural disas-
ters and this requires more cereals to be produced yearly
(Godfray et al. 2010; Wheeler and Braum 2013). Wheat
(Triticum aestivum L.) is an important food grain and
increasing its production may contribute to improved
global food security (Tester and Langridge 2010). It has
capacity during the grain filling period is associated with
increased grain yield (Masclaux-Daubresse et al. 2010;
Gregersen et al. 2013). There is a direct link between the
duration of flag leaf photosynthesis and cereal grain
yields (Gregersen and Krupinska 2014). Blake et al. (2007)
suggested that the duration of green leaves was posi-
tively correlated with yield and kernel weight. Gelang
et al. (2000) presented a highly positive correlation

Received 21 October 2014. Accepted 12 September 2015.

D. Yang, D. Peng, W. Yang, Y. Yin, Y. Li, and Z. Wang. State Key Laboratory of Crop Biology, College of Agronomy, Shandong
Agricultural University, Tai’an, Shandong 271018, People’s Republic of China.
Corresponding authors: Yong Li (woooowo@126.com); Zhen Wang (zlwang@sdau.edu.cn).

Can. J. Plant Sci. 96: 283–295 (2016) dx.doi.org/10.1139/cjps-2015-0368 Published at www.nrcresearchpress.com/cjps on 13 April 2016.
 284
between the grain-filling duration and flag leaf area
duration. However, with increasing leaf senescence, the
degradation of chlorophyll resulted in the decline in
photosynthetic capacity (Zhang et al. 2006). Therefore,
leaf senescence could be delayed or through stay-green
mechanisms that maintain green leaf area for longer
time, the yield of crop could be greatly improved
(Buchanan-Wollaston 1997).
Leaf senescence is a genetically programmed decline
in various cellular processes (Khanna-Chopra 2012).
Enhanced production of reactive oxygen species (ROS)
as well as malondialdehyde (MDA), an indicator of lipid
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peroxidation, is characteristic of senescent leaves
(Del Rio et al. 2006; Yarmolinskyet et al. 2014). Plant pos-
sess antioxidant defense mechanisms, comprising super-
oxide dismutase (SOD), peroxidase (POD), and catalase
(CAT) to avoid the deleterious effects of ROS and delay
leaf senescence (Gill and Tuteja 2010). It is reasonable
to hypothesize that increasing antioxidase enzyme
activities may delay leaf senescence and improve photo-
synthetic capacity. Stay-green crops have delayed chloro-
phyll degradation and longer green leaf duration
(Thomas and Howarth 2000). Stay-green mutants of
wheat are characterized by delayed senescence that have
For personal use only.
much higher antioxidant enzymes activities and grain
weight (Spano et al. 2003; Chen et al. 2010; Kusaba et al.
2013). The stay-green phenotype can be the result of
alterations in hormone metabolism and signaling,
particularly affecting networks involving cytokinins
and ethylene (Thomas and Ougham 2014). Stay-green cul-
tivar may have a defect in ethylene biosynthesis or sig-
naling (Shirzadian-Khorramabad et al. 2008) and show
enhanced contents of cytokinins and reduced levels of
ABA in leaves (He et al. 2005).
Timing of leaf senescence is controlled by the genetic
background (Cha et al. 2002), which is regulated by
several hormones (Graaff et al. 2006; Fukao et al. 2012).
It is anticipated that there are genes that link the cytoki-
nin response to leaf senescence (Lim et al. 2003).
Gibberellin acid (GA 3 ) decreased the activities of
enzymes involved in chlorophyll catabolism and
resulted in retarding senescence (Li et al. 2010). The role
of auxin in regulation of leaf senescence might be linked
with other hormones and metabolic flux (Schippers et al.
2007). The leaves of the ABA-treated wheat plants
remained green longer than the leaves of control plants
(Travaglia et al. 2010). ABA treatment resulted in H2O2
production in rice leaves, which preceded the occur-
rence of leaf senescence (Hung and Kao 2004). However,
ABA induced the expression of antioxidant genes and
enhanced the activities of antioxidative enzymes (Hung
and Kao 2003). In conclusion, although the effects of hor-
mones on plant growth have been reported, little is
known about exogenous ABA involved in regulating
both antioxidant enzymes and endosperm cell division
of winter wheat with different stay-green characteristics
after anthesis. Therefore, we conduct the experiments
Can. J. Plant Sci. Vol. 96, 2016
(1) to study the effects of ABA on grain filling for differ-
ent stay-green cultivars, (2) study the effects of ABA on
antioxidant enezyme activity, and (3) investigate the
respondence of endogenous hormone levels in grains to
exogenous ABA.
Materials and Method
Plant materials and growth condition
The field experiments were carried out in two growing
seasons from October 2010 to June 2011 and from
October 2011 to June 2012 at Tai’an Experimental
Station of Shandong Agricultural University, Tai’an,
China (36° 09’ N, 117° 09’ E, and 128 m above sea level).
Two wheat genotypes, stay-green Wennong 6 and the
control variety Jimai 20 (non-stay-green) were grown
in the experimental plots. The plot size was 9 m 2
(3 m × 3 m) with 10 rows (0.25 m between rows). The soil
contained 12.3 g kg−1 organic matter, 0.91 g kg−1 total N,
87.2 mg kg −1 available N, 8.6 mg kg −1 Olsen-P,
57.5 mg kg −1 Olsen-K. Before planting 120 kg ha −1 N,
75 kg ha−1 P2O5 and 150 kg ha−1 K2O were mixed into the
soil, and another 120 kg ha−1 N was applied at the joint-
ing stage. Seeds were sown on 10 October 2010 and
10 October 2011 with the density of 225 plants m−2. The
plants were harvested on 16 June 2011 and 14 June 2012,
respectively. Pests, diseases, and weeds were controlled
by appropriate chemical applications during crop cycle.
Experimental design
The experiment was 2 × 2 [two cultivars and two exog-
enous application (water and ABA)] in factorial design
with four treatments. Each of the treatments had three
plots as replicates in a complete randomized block
design. Date of anthesis (stage 65, Zadoks et al. 1974)
was determined when anthers shed at least 50% of the
spike. Distilled water or 10 mg L −1 of ABA (Sigma,
Chemical Co.) was sprayed at the rate of 100 mL m−2 on
the whole plants between 1 and 3 days after anthesis
(DAA) at 5:00 p.m. All the solutions contained Tween-20
at final concentrations of 0.5% (v/v) as a surfactant.
Sampling flag leaves
Uniform wheat plants flowering on the same day were
tagged for sampling. Thirty tagged flag leaves from each
treatment were sampled at 7 d intervals from anthesis to
35 DAA. Flag leaves were detached, immediately sub-
merged in liquid nitrogen for 0.5 h and then stored at
−40 °C until biochemical assays were performed.
Assays for antioxidase enzyme activities and malondialde-
hyde (MDA) content
Flag leaves (0.5 g) were crushed and homogenized with
5 mL 0.05 mol L−1 sodium phosphate buffer (pH 7.8) at
0 °C. The homogenate was centrifuged at 10 000g for
20 min, and the supernatants were used for enzyme
activity assays and malondialdehyde content determina-
tion. SOD activity was measured based on inhibition in
the photochemical reduction of nitroblue tetrazolium
Published by NRC Research Press
 Yang et al.
(NBT) following the method of Giannopolitis and Ries
(1977). The activity of SOD was measured by the rate at
which NBT was reduced by 50%, as measured by absorb-
ance at 560 nm. One unit of SOD was defined as the
amount of crude enzyme extract that is required for
inhibition the reduction rate of NBT by 50%. POD activity
was determined by the rate of guaiacol oxidation by H2O2
(Rao et al. 1995). The reaction mixture contained 100 mM
phosphate buffer (pH 6.0), 16 mM guaiacol, and 10 μl of
10% H 2 O 2, and 20 μl of the extract in a 3 ml volume.
POD activity was determined by the change in absorb-
ance at 470 nm per minute per gram fresh weight
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(ΔA470 g −1 min −1 FW). CAT activity was assayed in a reac-
tion mixture containing 2.0 ml 50 mM phosphate buffer
(pH 7.8), 0.5 ml 30 mM H2O2, and 0.1 ml of the extract in
a modified method following Aebi (1983). Unit of CAT
activity (ΔA240 g −1 min −1 FW) was defined as variation
of absorbance per minute per gram fresh weight. The
MDA content was measured according to Li (2000). 1 ml
of extract and 2 ml (0.6%, v/v) thiobarbituric acid were
boiled for 20 min and cooled to room temperature. The
absorbance of MDA was measured at 600, 532, and
450 nm after centrifuged at 3000g for 15 min. MDA
concentrations were calculated by the equation:
For personal use only.
MDA (μmol g −1 FW) = (6.45 × (OD 532 -OD 600 )-0.56 ×
OD 450 ) × V/W, where OD 532, OD 600 and OD 450 are the
absorbance at 532, 600, and 450 nm, respectively. V is
the volume of extraction. W is the fresh weight of sam-
ple. Three technical replicates were analyzed for each
sample.
Sampling grains
Three hundred spikes that flowered on the same day
were chosen and tagged for each experimental plot. 30
labeled spikes of each plot were sampled at 3, 6, 9, 12,
15, 18, 21, 24, 28, 35 DAA, respectively. Half-sampled
spikes were frozen in liquid nitrogen for 0.5 h and then
stored at −40 °C until biochemical assays were per-
formed. For dry weight determination another half-
sampled spikes were oven dried at 70 °C to constant
weight, dehulled, and weighted. From the basal 5–12
spikelets in the spikes of wheat, the first and second
basal grains on each spikelet were detached and consid-
ered as the superior grains. The third and fourth distal
grains were the inferior grains. These data were used to
simulate grain-filling process and calculate the grain-
filling rate.
The grain-filling process was fitted by the Richards
growth equation as described by Yang et al. (2001):
W = A=(1 þ Be−kt )1=N (1)
Grain-filling rate (G) was calculated as the derivative of
equation (1):
G = AkBe−kt =N(1 þ Be−kt )(Nþ1)=N (2)
285
Where W is the grain weight (g), A is the final grain
weight (g), t is the time after anthesis (d), and B, k, and
N are coefficients determined by regression.
Assays for endosperm cell number
The method for isolation and counting of endosperm
cell was according to Yang et al. (2002a). Ten superior or
inferior kernels were fixed in FAA solution (formalin:
acetic acid: ethyl alcohol (70%), 1:1:8, v/v) for 48 h.
Embryos were removed from these kernels, and were
stained with haematoxylin solution for 24 h, washed sev-
eral times with distilled water and then hydrolysed in
0.4% (w/v) cellulose at 40 °C for 8 h and oscillated.
Isolated endosperm cells were diluted to 10 mL. 1 mL sol-
ution was filtered through 0.45 μm hydrophobic mem-
branes, and a sample was placed on a glass slide with a
drop of glycerol. Using a light microscope, the endo-
sperm cell number of 10 fields of view for each mem-
brane was noted.
Determination of endogenous hormones in grains or flag
leaves
The extraction and purification of zeatin riboside
(ZR), gibberellin (GA 3 ), auxin (IAA), abscisic acid
(ABA) were measured by High Performance Liquid
Chromatography (HPLC), which was modified from
those previously described methods (Zhao et al. 2012).
1.0 gram grains or flag leaves were ground with liquid
nitrogen, added 4.0 mL of acetonitrile extraction
medium containing 30 mg L−1 sodium diethyldithiocar-
bamate as an antioxidant. The homogenate was incu-
bated in the dark at 4 °C for 12 h. The extracts were
centrifuged at 5000g for 15 min. The residue was further
extracted twice with the same solvent. The supernatant
was combined and concentrated to residue under
low pressure at 37 °C by rotatory evaporation, and
re-dissolved in 8.0 mL 0.4 mol L −1 phosphate buffer
(pH 8.0), and then added 6.0 mL chloroform and oscil-
lated to remove pigment. Chloroform phase was dis-
carded. 0.15 gram insoluble polyvinylpyrrolidone was
added into the aqueous phase to remove hydroxyben-
zene, and centrifuged at 10 000g for 10 min. 5.0 mL
supernatant was pipetted and adjusted to pH 3.0 with
pure formic acid. The aqueous phase was extracted
by 3.0 mL ethyl acetate two times. The ethyl acetate
phase was concentrated by rotatory evaporation under
low pressure, and re-dissolved in 1 mL mobile phase.
Finally, the hormone extraction was filtrated with
0.2 μm hydrophobic membranes, and then 10 μL samples
was injected into a Waters symmetry C18 column
(4.6 mm × 150 mm, 5 μm). Using acetonitrile: methanol:
0.6℅ acetic acid solution (5: 50: 45, V: V: V) as mobile
phase. The flow rate was kept at 0.6 mL min−1 and the
peaks were detected by a photodiode array detector
(Waters 2998 Separations Module, USA) absorbance at
254 nm.
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 286 Can. J. Plant Sci. Vol. 96, 2016

Table 1. Effects of exogenous abscisic acid (ABA) and genotype on activities of superoxide dismutase (SOD) in wheat leaves. The
mean square (MS) for the effects of hormone (ABA), varieties (V), and their interaction (V × ABA) are also shown.
Days after anthesis (DAA)

Cultivars Treatment 7 DAA 14 DAA 21 DAA 28 DAA 35 DAA

Jimai 20 Control 1241.68 ± 6.87 d 946.54 ± 9.31 d 490.56 ± 10.14 d 816.81 ± 3.35 d 35.9295 ± 2.02 d
ABA 1419.51 ± 12.26 c 1119.98 ± 12.25 c 653.50 ± 6.13 c 857.80 ± 6.02 c 116.77 ± 5.82 c
Wennong 6 Control 1521.22 ± 3.32 b 1171.97 ± 8.38 b 951.49 ± 20.11 b 1122.59 ± 15.83 b 159.26 ± 12.19 b
ABA 1596.77 ± 34.68 a 1438.93 ± 3.49 a 1338.07 ± 6.33 a 1463.44 ± 2.54 a 303.44 ± 7.57 a
MS(V) 156501.24** 222261.45** 984126.79** 623029.22** 72075.00**
MS(ABA) 48150.93** 145465.40** 226471.69** 109350.95** 37976.69**
MS(V × ABA) 7845.07** 6560.17** 37510.65** 67432.70** 3008.33**
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Note: Data represent means ± SE of 3 replicates. Different letters within the column indicate statistical at P < 0.05 level.
*, **denote levels of significance of MS values (0.05 and 0.01, respectively). One unit of SOD was defined as the amount of crude
enzyme extract that is required for inhibition the reduction rate of NBT by 50%.

Table 2. Effects of exogenous abscisic acid (ABA) and genotype on activities of peroxidase (POD, ΔA470 g−1 min−1 FW) in wheat
leaves. The mean square (MS) for the effects of hormone (ABA), varieties (V), and their interaction (V × ABA) are also shown.
Days after anthesis (DAA)

Cultivars Treatment 7 DAA 14 DAA 21 DAA 28 DAA 35 DAA

Jimai 20 Control 214.88 ± 3.11 d 165.65 ± 7.81 c 245.85 ± 3.61 d 188.14 ± 1.22 d 202.22 ± 4.32 c
ABA 288.85 ± 2.48 b 306.42 ± 3.83 a 286.42 ± 6.59 c 257.21 ± 3.65 b 206.34 ± 3.33 bc
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Wennong 6 Control 264.87 ± 3.10 c 285.54 ± 8.72 ab 338.55 ± 2.49 b 307.15 ± 3.39 a 2012.32 ± 1.89 b
ABA 318.85 ± 2.91 a 280.83 ± 3.56 b 365.95 ± 3.47 a 320.64 ± 2.77 a 231.50 ± 1.97 a
MS(V) 4800.01** 6668.12** 22246.06** 24963.66** 926.27**
MS(ABA) 12280.43** 13886.11** 3464.70** 5113.47** 403.31**
MS(V×ABA) 299.99** 15871.02** 130.07 2316.69** 172.45*
Note: Data represent means ± SE of 3 replicates. Different letters within the column indicate statistical at P < 0.05 level.
*, **denote levels of significance of MS values (0.05 and 0.01, respectively).

Table 3. Effects of exogenous abscisic acid (ABA) and genotype on activities of catalase (CAT, ΔA240 g−1 min−1 FW) in wheat
leaves. The mean square (MS) for the effects of hormone (ABA), varieties (V), and their interaction (V × ABA) are also shown.
Days after anthesis (DAA)

Cultivars Treatment 7 DAA 14 DAA 21 DAA 28 DAA 35 DAA

Jimai 20 Control 77.15 ± 0.16 c 62.59 ± 0.61 d 77.87 ± 0.62 a 40.77± 0.17 c 32.43 ± 0.18 c
ABA 91.25 ± 1.26 b 71.21 ± 0.59 c 74.52 ± 0.44 b 44.77 ± 0.18 a 37.77 ± 0.17 b
Wennong 6 Control 79.63 ± 0.93 c 84.63 ± 0.40 b 78.95 ± 0.46 a 42.69 ± 0.15 b 40.69 ± 0.45 a
ABA 105.365 ± 1.21 a 92.15 ± 0.51 a 75.80 ± 0.14 b 43.02 ± 0.30 b 31.68 ± 0.15 d
MS(V) 213.41** 1385.14** 4.13 0.02 1.02*
MS(ABA) 1207.42** 195.38** 31.77** 14.08** 16.33**
MS(V × ABA) 106.51** 0.90 0.04 10.08** 176.33**
Note: Data represent means ± SE of 3 replicates. Different letters within the column indicate statistical at P < 0.05 level.
*, **denote levels of significance of MS values (0.05 and 0.01, respectively).

Statistical analysis
The results were analyzed for variance and using
PASW software version 18.0. Data from each sampling
date were analyzed separately. The Duncan’s multiple
range test was used to determine the significance
of differences between treatments. The differences in
data across the two study years were not significant
(p > 0.05). Therefore, data for endogenous hormones con-
tents, endosperm cell division and grain filling were
from the growing season 2011–2012. The data for
enzymes activities, MDA content and grain yield were
averaged from the two years.
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 Yang et al. 287

Table 4. Effects of exogenous abscisic acid (ABA) and genotype on content of malonaldehyde (MDA, μmol g−1 FW) in
wheat leaves. The mean square (MS) for the effects of hormone (ABA), varieties (V), and their interaction (V × ABA) are
also shown.
Days after anthesis (DAA)

Cultivars Treatment 7 DAA 14 DAA 21 DAA 28 DAA 35 DAA

Jimai 20 Control 16.21 ± 0.13 a 30.49 ± 0.26 a 26.50 ± 0.27 a 40.44 ± 0.23 a 78.43 ± 0.84 a
ABA 13.33 ± 0.17 b 27.31 ± 0.51 b 24.48 ± 0.17 b 37.90 ± 0.33 b 66.90 ± 1.23 b
Wennong 6 Control 12.33 ± 0.81 b 27.11 ± 0.17 b 24.30 ± 0.53 b 32.40 ± 0.36 c 66.07 ± 2.15 b
ABA 10.34 ± 0.14 c 25.75 ± 0.38 c 119.91 ± 0.51 c 28.75 ± 0.60 d 58.75 ± 0.60 c
MS(V) 17.68** 18.16** 34.26** 221.35** 315.58**
MS(ABA) 35.30** 15.43** 30.79** 28.72** 266.64**
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MS(V × ABA) 0.58** 2.48** 4.23** 0.93* 13.33**

Note: Data represent means ± SE of 3 replicates. Different letters within the column indicate statistical at P < 0.05
level.
*, **denote levels of significance of MS values (0.05 and 0.01, respectively).

Fig. 1. Effects of exogenous ABA on content of four endogenous hormones in flag leaves. Control and ABA represent spraying
water and ABA, respectively. Symbols represent means ± standard error (n = 3).
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 288 Can. J. Plant Sci. Vol. 96, 2016

Fig. 2. Effects of exogenous ABA on content of four endogenous hormones in wheat kernels. The solid line and short dash line
represent superior kernels and inferior kernels, respectively. Closed circles and squares represent the control treatment. Open
circles and squares represent the exogenous ABA treatment. Symbols represent means ± standard error (n = 3). Vertical bars
indicate standard error.
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Results the decline of SOD activities was at a slower sequential

Antioxidant enzymes activities and MDA concentrations
analysis
Highly significant genotype × ABA interactions
(p < 0.01) were observed for SOD activities of flag leaves
(Table 1). There were significant differences in SOD activ-
ities of flag leaves between Wennong 6 and Jimai 20 after
anthesis. All SOD activities were higher in both control
and ABA treated Wennong 6 compared to Jimai 20.
Together, significant differences can be found between
ABA treated plants and the control plants. From 7 to 21
DAA SOD activities in ABA treated flag leaves of
Wennong 6 were maintained at the higher level, and
pace compared with the control samples. From 7 to
28 DAA POD activities of Jimai 20 were significantly
(p < 0.05) increased by application of ABA (Table 2),
meanwhile from 28 to 35 DAA CAT activities of Jimai 20
were significantly (p < 0.05) increased (Table 3). Highly
significant genotype × ABA interactions (p < 0.01) were
observed for CAT activities of flag leaves at 7, 28, and
35 DAA.
There were highly significant (p < 0.01) genotype ×
ABA interactions for MDA concentrations of flag leaves
at 7, 14, 21 and 35 DAA (Table 4). MDA concentrations of
flag leaves in Wennong 6 from the control samples were
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 Yang et al.
significantly (p < 0.05) lower than those of Jimai 20. MDA
concentrations of flag leaves in both cultivars were sig-
nificantly decreased by application of exogenous ABA.
For example, Wennong 6 showed 11.1% reduction in
MDA concentrations at 35 DAA. In contrast, Jimai 20
showed 14.7% reduction at 35 DAA.
Changes in the hormonal levels in the flag leaves
The ZR content in stay green wheat Wennong 6 was
significantly (p < 0.05) higher than those in Jimai 20 on
days 7 and 14. Treatment with exogenous ABA signifi-
cantly (p < 0.05) decreased the levels of endogenous ZR
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from 7 to 28 DAA, compared with the control leaves of
both cultivars (Fig. 1A and B).
Endogenous ABA levels of all treatments increased
firstly and peaked at 21 DAA, thereafter, it declined
gradually (Fig. 1C and D). ABA contents in flag leaves of
Wennong 6 were lower than those in flag leaves of
Jimai 20 especially at 21 to 35 DAA. Spraying with ABA
significantly increased endogenous ABA levels in flag
leaves of Wennong 6 from 7 to 21 DAA, but significantly
decreased ABA levels in flag leaves of Jimai 20 from 21
to 35 DAA.
Endogenous GA3 reached a maximum value for con-
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trol Wennong 6 and Jiami 20 at 14 and 28, respectively.
(Fig. 1E and F). Although exogenous ABA decreased
the GA3 levels in flag leaves of Jimai 20 at 7 DAA, the con-
tent of GA3 in Jimai 20 significantly increased after ABA
treatment from 14 to 21 DAA and reaching a peak at
14 DAA.
The IAA contents of both cultivars increased after
anthesis and peaked at 21 DAA for all the treatments,
thereafter, it declined gradually (Fig. 1G and H). From 7
to 28 DAA the IAA levels in flag leaves of two cultivars
were significantly increased by spraying with ABA.
Changes in the hormonal levels in grains
The levels of the four endogenous hormones
responded differently to exogenous ABA applied at dif-
ferent stages (Fig. 2). ZR contents in the superior grains
of the two cultivars transiently increased at early grain
filling stage, and reached a small peak at 6 DAA, then
increased and reached the maximum content at 18
DAA, and decreased thereafter (Fig. 2A and B). ZR con-
tents in the inferior grains of the two cultivars increased
from 6 to 18 DAA, and reached a maximum at 18 DAA,
and decreased thereafter. Exogenous ABA obviously
decreased the ZR contents of superior grains in the two
cultivars at 15 to 18 DAA.
Endogenous ABA content in superior grains of both
cultivars increased slowly at 3 to 6 DAA, and markedly
increased from 15 to 21 DAA for superior grains of Jimai
20 and 9 to 21 DAA for the superior of Wennong 6
(Fig. 2C and D). ABA content in superior grains of Jimai
20 reached a maximum at 21 DAA under Control and
ABA treatment, while ABA in inferior grains of Jimai 20
reached a maximum at 15 DAA under Control and ABA
289
Table 5. Effects of exogenous abscisic acid (ABA) and
genotype on 1000-grain weight (g) and grain yield (g m−2).
The mean square (MS) for the effects of hormone (ABA),
varieties (V), and their interaction (V × ABA) are also shown.
1000-grain Grain yield
Cultivars Treatment weight (g) (g m−2)
Jimai 20 Control 32.75 ± 0.07 d 654.10 ± 1.92 d
ABA 35.28 ± 0.19 c 689.17 ± 3.02 c
Wennong 6 Control 39.88 ± 0.12 b 738.69 ± 9.88 b
ABA 42.44 ± 0.31 a 818.78 ± 5.31 a
MS(V) 175.14** 68821.74**
MS(ABA) 24.31** 19893.12**
MS(V×ABA) 1.03** 3040.50**
Note: Data represent means ± SE of 3 replicates. Different
letters within the column indicate statistical at P < 0.05
level.
*, **denote levels of significance of MS values (0.05 and
0.01, respectively).
treatment. Spraying with ABA significantly increased
endogenous ABA levels in the superior grains of both
cultivars at 18 to 24 DAA. ABA levels in the inferior grains
of both cultivars were significantly increased by ABA
treatment at 15 to 18 DAA.
GA 3 contents in the superior and inferior grains of
Jimai 20 were high at early grain filling stage, and
reached a maximum at 3 DAA for superior grains and
6 DAA for inferior grains under Control and ABA treat-
ment (Fig. 2E and F). While the maximum GA3 contents
in superior grains of Wennong 6 were at 6 DAA and in
inferior grains of Wennong 6 were at 18 DAA under
Control and ABA treatment. GA3 contents in superior
grains were decreased dramatically by exogenous ABA
at 3 to 9 DAA and 18 to 24 DAA. GA3 contents in inferior
grains of Wennong 6 were significantly decreased by
ABA treatment at 9 to 24 DAA.
IAA content in the grains was low and slowly changed
at 3 to 9 DAA, but markedly increased from 9 to 18 DAA
and reached a maximum at 18 DAA under Control and
ABA treatments (Fig. 2G and H). Application of ABA sig-
nificantly enhanced IAA levels in the superior grains of
Jimai 20 at 12 to 18 DAA. Exogenous ABA increased IAA
levels in the inferior grains of Wennong 6 at 6 and
12 DAA.
Grain weight, grain filling, and endosperm cell division
Exogenous ABA significantly (p < 0.01) increased 1000-
grain weight and grain yield in two cultivars (Table 5).
Figure 3 showed the grain filling progress and grain-
filling rate in both superior and inferior grains. Both cul-
tivars showed a faster grain-filling rate and higher grain
weight in superior grains than in inferior grains. There
were obvious differences in the grain weight and filling
rate between Wennong 6 and Jimai 20. Application of
ABA significantly improved grain weight and accelarated
the grain-filling rate. For example, at 35 DAA the weight
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 290
Fig. 3. Grain filling process (A, B) and grain-filling rate (C, D) of Jimai 20 and Wennong 6. The circles and triangles represent
superior and inferior grains, respectively. The solid circles and triangles represent ABA treatment. The hollow circles and
triangles represent control treatment. Symbols represent means ± standard error (n = 3). Vertical bars indicate standard error.
Can. J. Plant Sci. Downloaded from www.nrcresearchpress.com by 180.251.158.61 on 03/23/18
For personal use only.
of superior grains and inferior grains in ABA-treated
Wennong 6 were about 5.39 and 5.84% higher than the
control treatments, respectively. In contrast, the supe-
rior grains weight and inferior kernel weight of Jimai
20 were increased by 15.01 and 14.44%, compared to the
control treatment, respectively.
Similarly to the changing pattern of grain weight,
endosperm cell number was higher in the superior
grains than in inferior grains (Fig. 4). The superior grains
also had much higher endosperm cell division rate than
the inferior grains. The endosperm cell number of
ABA-treated plants was significantly improved. The
endosperm cell division rate of both superior and
inferior grains of jimai 20 were improved by application
of ABA from 3 to 15 DAA. The endosperm cell division
rate of inferior grains in Wennong 6 was also increased
by exogenous ABA from 6 to 24 DAA.
Relationships between hormone contents in grains and
grain-filling rate
The ZR, ABA and IAA content of grains were positively
and significantly correlated with grain-filling rate at 3 to
15 DAA and 18 to 27 DAA, respectively (Fig. 5). GA3 con-
tent in grains of Jimai 20 was negatively and significantly
with grain-filling rate at 3 to 15 DAA, but significantly
and positively correlated with grain-filling rate at 18 to
27 DAA. Together, only at 18 to 27 DAA GA3 content in
Can. J. Plant Sci. Vol. 96, 2016
grains of Wennong 6 showed significantly and positively
correlated with grain-filling rate.
Relationships between hormone contents in leaves and
grain-filling rate
In this study, it was found that ZR content in leaves of
Jimai 20 was negatively and significantly correlated with
grain-filling rate at 7 to 35 DAA (Fig. 6). GA3 content in
leaves of Wennong 6 was positively and significantly cor-
related with grain-filling rate. IAA contents in leaves of
both cultivars were positively and significantly corre-
lated with grain-filling rate.
Discussion and Conclusions
Leaf senescence is an extremely complex physiological
and biochemical process and is the final stage of leaf
development (Morris et al. 2000; Chandlee 2001). It
involves degradation of chlorophyll and increase in reac-
tive oxygen species (ROS) and lipid peroxidation with the
concomitant production of MDA (Navabpour et al. 2003;
Pruzinska et al. 2005), which acts as an estimator of
the degree of oxidative stress experienced by the tissue
(Hodges et al. 1999). SOD, POD and CAT play important
roles in avoiding the deleterious effect of ROS
(Djanaguiraman et al. 2009). Stay-green mutants have
been reported to maintain leaf greenness longer than
their wild types during senescence (Hortensteiner
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 Yang et al.
Fig. 4. Endosperm cell number (A, B) and endosperm cell division rate (C, D) of Jimai 20 and Wennong 6. The circles and triangles
represent superior and inferior grains, respectively. The solid circles and triangles represent ABA treatment. The hollow circles
and triangles represent control treatment. Symbols represent means ± standard error (n = 3). Vertical bars indicate standard error.
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For personal use only.
2009). In stay-green maize, the delay of senescence was
associated with higher CAT and SOD levels (He et al.
2005). Hui et al. (2012) suggest that the higher antioxi-
dant defense system may be one of the most important
mechanisms underlying the expression of the delayed
senescence phenotype in staygreen wheat. In this study,
staygreen wheat (Wennong 6) exhibited the higher
SOD, POD, and CAT activity and lower MDA content than
those in Jimai 20. Endogenous hormones may play
important roles. The ZR content in Wennong 6 was
higher than those in Jimai 20 on days 7 and 14. ABA con-
tents in Wennong 6 were lower than those in Jimai 20 at
21 to 35 DAA. In the present study, although the endog-
enous ZR content of flag leaves was decreased with
spraying exogenous ABA from 7 to 28 DAA, IAA content
of ABA-treated plants was enhanced at 7 to 28 DAA. IAA
delayed leaf senescence because they were related to
inhibit activities of proteolytic enzymes and RNase
(Xiao et al. 1998). Similar studies showed that IAA inhib-
ited the expression of senescence associated gene and
reduced accumulation of 1-amino cyclopropane carbox-
ylic acid, thereby reduced the biosynthesis of ethylene
(Bao et al. 2001). IAA could improve pigment production
and delayed leaf senescence (Aldesuquy 2000). Although
it has been reported that ABA accelerated leaf senes-
cence (Hung and Kao 2004), molecular genetic analysis
291
indicated that there was not a crucial link between ABA
and senescence (Fedoroff 2002; Jing et al. 2003). It has
been reported that exogenous ABA directly or indirectly
induced the expression of SOD genes such as Fe-SOD
gene and the other cytosolic Cu/Zn-SOD gene (Xu et al.
2009), and enhanced the capacity of antioxidant defence
systems in plants (Jiang and Zhang 2004). The results
reported here suggest that the MDA contents in flag
leaves from the control samples were higher than the
ABA-treated plants in both cultivars. SOD, CAT and POD
activities in flag leaves from the ABA-treated plants were
significantly higher than those from the control plants.
These suggest that low concentrations of exogenous
ABA (10 mg L−1) improved the antioxidant capacity of flag
leaves and reduced membrane lipid peroxidation with
decreasing MDA concentration.
Plant hormones play important roles in mediating
nutrient allocation (Peleg and Blumwald 2011). IAA at a
high concentration in grains improved photosynthetic
assimilations remobilization and enhanced the grain fill-
ing (Yan et al. 2005). There was significantly positive cor-
relation between IAA content and grain filling rate
(Xu et al. 2013). Jiang et al. (2006) reported that ABA accel-
erated the sucrose accumulation in grains, this may be
due to the improved absorption and unload of sucrose
in grain sink (Jones and Brenner 1987). ABA increased
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 292 Can. J. Plant Sci. Vol. 96, 2016

Fig. 5. Relationship between grain filling rate and the hormone concentrations in the kernels. Closed triangles and open circles
represent hormone concentrations at 3 to 15 days after anthesis (DAA) and at 18 to 28 DAA, respectively. The solid line and the
short dash line represent the regression line between grain-filling rate and hormone concentrations at 3 to 15 DAA (n = 18) and at
18 to 28 DAA (n = 16), respectively. The hormone concentrations and grain filling rates that were used in the analysis were pooled
from 9 time points, inferior and superior grains and ABA or water treated plants. Each data point is the mean of 3 replications.
r represents correlation coefficient. Asterisks “*” and “**” represent significance at 0.05 and 0.01 probability level, respectively.
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For personal use only.

carbon remobilization and accumulation in wheat
grains (Yang et al. 2002b; Saeedipour and Moradi 2012).
In the present study, although ZR contents of grains
were decreased by exogenous ABA, endogenous ABA lev-
els in the superior and inferior grains of both cultivars
were increased by application ABA at 18 to 24 DAA and
15 to 18 DAA, respectively. IAA levels in the inferior grains
of Wennong 6 were increased by ABA at 6 and 12 DAA.
We found that exogenous ABA increased grain-filling rate
and endosperm cell division rate, and finally increased
endosperm cell number and improved grain weight.
Hence, on basis of our results and those of previous
studies, we can summarize that the increase of grain
weight under exogenous ABA treatment were closely
associated with (1) the changed levels of endogenous hor-
mones in flag leaves that induced increase of antioxidant
enzyme activities and decrease of MDA accumulation,
which delayed leaf senescence to facilitate more photo-
synthate formation and remobilization; (2) the changed
hormones levels in grains that enlarged endosperm cell
number and accelerated the grain-filling rate, which
facilitated more photosynthate accumulation in grains.
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 Yang et al. 293

Fig. 6. Relationship between grain filling rate and the hormone concentrations in flag leaves. The solid line represents the
regression line between grain-filling rate and hormone concentrations at 7 to 35 DAA (n = 20). Each data point is the mean of
3 replications. r represents correlation coefficient. Asterisks “*” and “**” represent significance at 0.05 and 0.01 probability level,
respectively.
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For personal use only.

Acknowledgments
The research was supported by the National Natural
Science Foundation of China (NO. 31271661, 30871477),
the Shandong Modern Agriculture Technology and
Industry System. The National Basic Research Program
of China (973 Program, NO. 2009CB118602), the Special
Fund for Agro-scientific Research in the Public Interest
of China (No. 201203100, 201203029), and the National
Science and Technology Support Program of China
(No. 2012BAD04B05).
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