全 文 ：蛋白激酶组在玉米叶片 ABA和 H2O2 诱导
抗氧化防护中的作用*
许树成1, 祝雪兰2**, 张摇 丽3**
(1 阜阳师范学院生命科学学院, 安徽 阜阳摇 236041; 2 安徽阜阳市红旗中学, 安徽 阜阳摇 236034;
3 安徽省金寨县南溪中学, 安徽 六安摇 237321)
摘要: 蛋白磷酸化在植物细胞脱落酸 (ABA) 介导的信号转导中起重要作用。 然而, 很多参与 ABA 信号
途径的蛋白元件仍不清楚。 使用改进的体外激酶试验方法的研究结果表明, 在玉米叶片中, ABA和 H2O2
能够快速活化蛋白激酶总活性和 Ca2+依赖型蛋白激酶总活性; ABA 诱导的蛋白激酶总活性增加可以被活
性氧的抑制剂和清除剂抑制, 蛋白激酶抑制剂不仅可以降低 ABA和 H2O2诱导的激酶活性增加, 而且也可
以弱化它们对抗氧化防护酶活性的诱导作用; ABA和 H2O2引发的蛋白磷酸化作用显著居先于它们诱导的
抗氧化防护作用。 使用凝胶激酶试验方法进行研究发现, 一组分子量分别为 66 kDa, 52 kDa, 49 kDa和 35
kDa 的蛋白激酶可能介导了 ABA和 H2O2诱导的抗氧化防护反应, 并且 66 kDa和 49 kDa的蛋白激酶可能在
ROS的下游起作用, 而 52 kDa和 35 kDa的蛋白激酶可能在 ABA和 ROS的下游起作用。
关键词: ABA; 蛋白激酶; 磷酸化作用; 活性氧; 抗氧化防护
中图分类号: Q 945摇 摇 摇 摇 摇 摇 文献标识码: A摇 摇 摇 摇 摇 摇 摇 摇 文章编号: 2095-0845(2011)03-275-12
Role of Protein Kinases in Abscisic Acid and H2O2
Induced Antioxidant Defense in Maize
XU Shu鄄Cheng1, ZHU Xue鄄Lan2**, ZHANG Li3**
(1 School of Life Sciences, Fuyang Teachers College, Fuyang 236041, China; 2 Hongqi Middle School,
Fuyang 236034, China; 3 Nanxi Middle School, Lu忆an 237321, China)
Abstract: Protein phosphorylation plays a central role in mediating abscisic acid (ABA) signaling transduction in
plant cells, whereas many of the sensory proteins involving in ABA signaling pathway remain unclear. Here, using a
modified in vitro kinase assay, our results showed that ABA and H2O2 induced a rapid activation of total protein ki鄄
nases and calcium dependent protein kinases in the leaves of maize seedlings. However, ABA鄄induced activation of
protein kinases was inhibited by reactive oxygen species (ROS) inhibitors or scavengers. Protein kinase inhibitors
decelerated not only the ABA and H2O2 鄄induced kinase activity but also ABA or H2O2 鄄induced antioxidant enzyme
activity. Protein phosphorylation caused by ABA and H2O2 preceded ABA or H2O2 鄄induced antioxidant defense ob鄄
viously. Using in鄄gel kinase assays, our results showed that several protein kinases with molecular masses of 66
kDa, 52 kDa, 49 kDa and 35 kDa respectively might mediate ABA and H2O2 鄄induced antioxidant defense. And the
66 kDa and 49 kDa protein kinases may act downstream of ROS, and the 52 kDa and 35 kDa protein kinases may act
植 物 分 类 与 资 源 学 报摇 2011, 33 (3): 275 ~ 286
Plant Diversity and Resources摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 DOI: 10. 3724 / SP. J. 1143. 2011. 10197
*
**
Foundation items: The study project of Anhui Education Department (KJ2010B156 ), the Natural Science Foundation of AnHui Province
(11040606M86)
与第一作者同等贡献 (The author contributed equally to this work)
Received date: 2010-11-06, Accepted date: 2011-02-21
作者简介: 许树成 (1969-) 男, 理学博士, 教授, 主要从事植物逆境细胞信号转导研究以及细胞生物学和遗传学教学与科
研。 E鄄mail: xscjack@ 126. com
between ABA and ROS in ABA鄄induced antioxidant defensive signaling.
Key words: Abscisic acid ; Protein kinase; Phosphorylation; Reactive oxygen species; Antioxidant defense
摇 摇 Plants have to confront various biotic and abiotic
stresses during their growth and development. To sur鄄
vive, plants have evolved intricate mechanisms to a鄄
dapt to environmental changes. The molecular and bio鄄
chemical aspects of how plants perceive and respond to
environmental signals are not clearly understood.
One important regulator of plant responses to
abiotic stresses is the phytohormone ABA. ABA
plays an important role in response to abiotic stresses
such drought, low temperature, high temperature
and salinity (Finkelstein et al., 2002; Zhang et al.,
2009). Recently, the increasing acuumulation of
evidences reveal the importance of ABA in response
to oxidative stress in plant cells. ABA can cause the
generation of ROS in various plant cells or tissues,
induce the expression of antioxidant genes, and en鄄
hance the capacity of antioxidant defense systems,
including enzymatic and nonenzymatic constituents
(Guan et al., 2000; Pei et al., 2000; Jiang and
Zhang, 2001; Zhang et al., 2006; Hu et al.,
2005). However, the mechanisms that ABA鄄in鄄
duced ROS production up鄄regulates antioxidant de鄄
fense have yet to be determined.
Evidences from biochemical and genetic studies
of plant stress signaling indicate that reversible pro鄄
tein phosphorylation plays an important role in the
regulation of physiological status and gene expression
in response to various environmental stresses (Yuasa
et al., 2001; Xiong and Yang, 2003). So it is of
special importance to study protein phosphorylation
in ABA鄄induced antioxidant defense.
As an important calcium sensor, Ca2+ 鄄depend鄄
ent protein kinases (CDPKs) play important roles in
response to various abiotic stresses ( Klimecka and
Muszynska, 2007). Potata StCDPK4 and StCDPK5
activate StRBOHB protein by phosphorylation of N
terminus region in a calcium鄄dependent manner
(Kobayashi et al., 2007). These results indicate
that CDPKs play important roles in regulating the pro鄄
duction of ROS and may act upstream of H2O2 signa鄄
ling under stress. However the snf1鄄related kinase
OST1 act in the upstream of H2O2 signaling in a cal鄄
cium鄄independent manner (Mustilli et al., 2002).
It has been documented that ABA can induce
the production of ROS and the increase of cytosolic
Ca2+ ( [Ca2+] cyt) (Pei et al., 2000; Kwak et al.,
2003). H2O2 treatment also induces [Ca2+] cyt ele鄄
vations (Rentel and Knight, 2004) and the expres鄄
sion of the CaM gene (Desikan et al., 2001). Usu鄄
ally, CaM has no enzymatic activity, the active
Ca2+ 鄄CaM complex regulates the activity of down鄄
stream target proteins (Snedden and Fromm, 2001;
McCormack et al., 2005). Transgenic tobacco ex鄄
pressing a foreign CaM gene showed an enhanced
production of ROS (Harding et al., 1997). Extra鄄
cellular CaM stimulates an intracellular signaling
pathway involving activation of a heterotrimeric G
protein, H2O2 generation, and changes in [Ca2+] cyt
in the regulation of stomatal movements ( Chen et
al., 2004). Calcium / CaM plays a critical role in
controlling H2 O2 homeostasis in plants ( Yang and
Poovaiah, 2002). In Arabidopsis thaliana, the ex鄄
pression of calcium / CaM鄄regulated kinase, CRCK1
is stimulated by cold, salt, ABA, and H2O2 treat鄄
ments ( Yang et al., 2004). These studies imply
that not only CaMs but also CaMKs are involved in
ROS signaling in plants.
However, it is not clear whether CDPK and
CaMK pathways are involved in ABA鄄enhanced an鄄
tioxidant defense systems in plants. Moreover, both
ABA and H2 O2 can activate some protein kinases,
suggesting that ABA and H2O2 may converge on pro鄄
tein phosphorylation signaling pathways in regulation
of stomatal movement ( Desikan et al., 2004 ).
However the relationship between ABA, protein ki鄄
nase, and H2O2 production remains to be determined
in ABA signaling. Here our results reported that the
calcium dependent protein kinase and some other
672摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 植 物 分 类 与 资 源 学 报摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 第 33 卷
protein kinase activities were detected and character鄄
ised in response to ABA and H2O2 .
Phosphorylation by protein kinases is crucial to
many cellular processes. Thus, assays to identify or
characterize kinases are of a key tool in this area. In
this study, an effort was made to elucidate whether
some protein kinases are involved in ABA or H2O2
signaling. And the relationship between ABA, H2
O2, kinases, and antioxidant defensive system was
also discussed.
1摇 Materials and methods
1. 1摇 Plant materials and treatments
Seeds of maize (Zea mays L., cv. Nongda 108;
from Nanjing Agricultural University, China) were
sown in trays of sand in a light chamber at a temper鄄
ature of 22益 to 28益, with a photosynthetic active
radiation of 200 滋mol m-2 s-1 and a photoperiod of
14 / 10 h (day / night), and watered daily.
When the second leaf was fully expanded, the
plants were collected and used for all investigations.
The plants were excised at the base of the stem, and
placed in the distilled water for 1 h to eliminate the
wound stress. After the treatment, the cut ends of the
stems were placed in the beakers wrapped with alumi鄄
num foil containing 100 滋mol ABA or 10 mmol·L-1
H2O2 solution for 12 h at 25益, with a continuous
light intensity of 200 滋mol m-2s-1 .
To study the effects of inhibitors and scaven鄄
gers, the detached plants were pretreated with 1 滋mol
K252a, 10 滋mol·L-1 H鄄7, 1 滋mol·L-1 staurospo鄄
rine, 10 mmol·L-1 Tiron and 5 mmol·L-1 DMTU for
4 h and then exposed to ABA or H2O2 treatment for
0-12 h under the same conditions as described.
Detached plants were treated with distilled water
under the same conditions for the whole period and
served as controls for the above. After treatments of
detached maize plants, the second leaves were sam鄄
pled and immediately frozen under liquid N2 .
1. 2摇 In vitro kinase activity assays
Kinase activity was analyzed in solution essen鄄
tially as described (Yu et al., 2006). Protein was
extracted from maize leaves (2 g) with 2 volumes
(w / v) of extraction buffer (50 mmol·L-1 Tris, pH
7. 5, 5 mmol·L-1 EDTA, 5 mmol·L-1 EGTA, 10
mmol·L-1 DTT, 10 mmol·L-1 Na3V04, 10 mmol·
L-1 NaF, 50 mmol·L-1 茁鄄glycerophosphate, 1 mmol·
L-1 PMSF, 5 滋g·mL-1 antipain, 5 滋g·mL-1 aprotinin,
5 滋g·mL-1 leupeptin, 10% glycerol). After centrifu鄄
gation at 15 000 g for 45 min at 4益, the supernatants
with crude proteins were used for kinase assays. Pro鄄
tein concentration was determined by the Bradford
method with bovine serum albumin as standard.
To assay kinase activity, kinase activity was de鄄
termined in 50 滋L reaction buffer containing 10 滋g of
total protein from extracts, 0. 5 mg·mL-1 histone III鄄S,
50 mmol·L-1 Tris, pH 7. 5, 5 mmol·L-1 MgCI2, 1
mmol·L-1 EGTA (or 2 mmol·L-1 CaCl2 when nee鄄
ded), 1 mmol·L-1 DTT. The phosphorylation reaction
was initiated by addition of 25 滋mol·L-1 ATP contai鄄
ning 50 滋Ci mL-1 [酌鄄32 P] ATP. The reaction was
carried out at 25益 for 20 min and terminated by trans鄄
ferring 20 滋L reaction mixture to P81 filter treated by
TCA. After an extensive wash in 5% TCA solution at
4益, P81 filter was dehydrated in ethanol and air
dried. The bound radioactivity in the P81 filter was
measured with a liquid scintillation counter.
1. 3摇 In鄄gel protein kinase assays
In鄄gel kinase activity assays were performed es鄄
sentially as described previously ( Zhang and Kles鄄
sig, 1997). Extracts containing 10 滋g of protein
were electrophoresed on 10% SDS鄄polyacrylamide
gels embedded with 0. 25 mg·mL-1 histone III鄄S in
the separating gel as a substrate for the kinase as鄄
say. After electrophoresis, SDS was removed by
washing the gel with washing buffer (25 mmol·L-1
Tris, pH 7. 5, 0. 5 mmol·L-1 DTT, 0. 1 mmol·L-1
Na3V04, 5 mmol·L-1 NaF, 0. 5 mg mL-1 BSA, 0. 1%
Triton X鄄100 [v / v]) three times, each for 30 min
at room temperature. The kinases were allowed to
renature in 25 mmol·L-1 Tris, pH 7. 5, 1 mmol·
L-1 DTT, 0. 1 mmol·L-1 Na3V04, and 5 mmol·L-1
NaF at 4益 overnight with three changes of buffer.
The gel was then incubated at room temperature in a
7723 期摇 摇 XU and ZHU: Role of Protein Kinases in Abscisic Acid and H2O2 Induced Antioxidant Defense in Maize摇 摇 摇
30 mL reaction buffer (25 mmol·L-1 Tris, pH 7. 5,
2 mmol·L-1 EGTA or no EGTA but 2 mmol·L-1
CaCl2, 14 滋g ml-1 CaM were added to reaction buff鄄
er. when needed, 12 mmol·L-1 MgCl2, 1 mmol·L-1
DTT, 0. 1 mmol·L-1 Na3V04) with 250 nmol ATP
containing 50 滋Ci mL-1 for 60 min.
The gels were then washed extensively with 5%
trichloroacetic acid ( TCA) and 1% sodium pyro鄄
phosphate until radioactivity in the used wash solu鄄
tion was barely detectable. The gels were then
stained with Coomassie Brilliant Blue R鄄250. After
destaining, the gels were air dried between two
sheets of cellophane and was detected by autoradio鄄
graphy after exposition of the dried gels to Kodak X鄄
Omat AR film for 2 to 3 d at -20益 . Prestained size
markers (Bio鄄Rad) were used to calculate the size
of kinases.
Quantitation of the relative kinase activities was
done using a PhosphorImager. Autophosphorylation
of protein activity assay was performed as described
above, except that the separating gel was not poly鄄
merized in the presence of 0. 25 mg mL-1 histone III鄄
S as a substrate for kinases.
1. 4摇 Antioxidant defensive enzyme assays
Frozen leaf segments (1 g) were homogenized
in 10 mL of 50 mmol·L-1 potassium phosphate buff鄄
er, pH 7. 0, containing 1 mmol·L-1 EDTA and 1%
polyvinylpyrrolidone, with the addition of 1 mmol·
L-1 ascorbate in the case of APX assay. The homoge鄄
nate was centrifuged at 15 000 g for 20 min at 4益
and the supernatant was immediately used for the
following antioxidant enzyme assays. Protein concen鄄
tration was determined by the Bradford method with
bovine serum albumin as standard.
The total activities of antioxidant enzymes were
determined as described previously ( Jiang and
Zhang, 2002b).
Total SOD ( superoxide dismutase ) activity
was assayed by minitoring the inhibition of photo鄄
chemical reduction of NBT (nitro blue tetrazolium).
The 3 mL reaction mixture contained 50 mmol·L-1
potassium phosphate buffer ( pH 7. 8), 13 mmol·
L-1 methionine, 75 滋mol·L-1 NBT, 2 滋mol·L-1 ri鄄
boflavin, 0. 1 mmol·L-1 EDTA, and 100 滋L enzyme
extract. The reaction mixtures were illuminated for
15 min at a light intensity of 400 滋mol·m-2·s-1 .
One unit of SOD activity was defined as the amount
of enzyme required to cause 50% inhibition of the
reduction of NBT as monitored at 560 nm.
CAT (catalase) activity was determined by fol鄄
lowing the consumption of H2O2 ( extinction coeffi鄄
cient 39. 4 mM-1 cm-1) at 240 nm for 3 min. The re鄄
action mixture contained 50 mmol·L-1 potassium
phosphate buffer ( pH 7. 0), 10 mmol·L-1 H2 O2
and 200 滋L of enzyme extract in a 3 mL volume.
APX ( ascorbate peroxidase) activity was de鄄
termined by following the decrease in A290 (extinc鄄
tion coefficient 2. 8 mM-1 cm-1) for 1 min in 1 mL of
reaction mixture containing 50 mmol·L-1 potassium
phosphate buffer ( pH 7. 0), 0. 5 mmol·L-1 ASC
(ascorbic acid), 0. 1 mmol·L-1 H2O2, and 200 滋L
of enzyme extract. The reaction was started by en鄄
zyme extract. Correction was done for the low, non鄄
enzymatic oxidation of ASC by H2O2 .
GR (glutathione reductase) activity was deter鄄
mined by following the oxidation of NADPH at 340
nm (extinction coefficient 6. 2mM-1 cm-1) for 3 min
in 1 mL of an assay mixture containing 50 mmol·L-1
potassium phosphate buffer (pH 7. 8), 2 mmol·L-1
EDTA, 0. 15 mmol·L-1 NADPH, 0. 5 mmol·L-1
GSSG (Oxidized glutathione), and 100 滋L of en鄄
zyme extract. The reaction was initiated by adding
NAPDH. Corrections were made for the background
absorbance at 340 nm, without NADPH.
2摇 Results
2. 1摇 Effects of ABA and H2O2 on the activities
of total protein kinases and calcium dependent
protein kinases
Previous studies have shown that protein revers鄄
ible phosphorylation events play important roles in
ABA signaling in guard cells (Li et al., 2000; Mer鄄
lot et al., 2001; Kwak et al., 2002; Mustilli et al.,
2002). It has also been shown that both ABA and
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H2O2 can activate the same MAPK ( Lu et al.,
2002; Desikan et al., 2004), and ROS are required
for the ABA鄄induced antioxidant defense (Hu et al.,
2005; Jiang and Zhang, 2002a, b).
To investigate the effects of ABA and H2O2 on
the protein phosphorylation level in maize plants, in
vitro kinase assays with histome III鄄S as a substrate
were determined. Treatment with 100 滋mol·L-1
ABA or 10 mmol·L-1 H2O2 increased the total pro鄄
tein kinase activity within 20 min, maximized at 60
min, remained high for 90 min after ABA treatment,
and then decreased after 180 min of ABA or H2O2
treatment (Fig. 1: A). After treatment with ABA for
60 min, total protein phosphorylation level was in鄄
creased by 97. 3% compared with the control, wher鄄
eas H2O2 treatment only caused 67. 1% phosphoryl鄄
ation level increase. Treatment with ABA or H2 O2
also led to a significant increase in the activity of
calcium dependent protein kinases ( Fig. 1: B).
When treated with ABA or H2O2 for 30 min, the cal鄄
cium dependent protein kinase activity reached the
maximum value and enhanced by 136. 8% , 84. 2%
respectively compared with the control. These results
indicated that the ABA and H2O2 induce an earlier
and stronger response in terms of calcium dependent
protein kinases activity.
Fig. 1摇 Time course of changes in the activities of total protein kinase activities and Ca2+ dependent protein kinase activities in maize
leaves treated by ABA or H2O2 . (A) Effects of ABA and H2O2 on the total protein kinase activities. (B) Effects of ABA and
H2O2 on the Ca2+ dependent kinase activities. (C) Effects of pretreatment with H2O2 scavengers DMTU, Tiron on the total protein
kinase activities induced by ABA and H2O2 . (D) Effects of pretreatment with H2O2 scavengers DMTU, Tiron on the Ca2+ depen鄄
dent kinase activities induced by ABA and H2O2 . The values are the mean依SE (n=6) for three different experiments
9723 期摇 摇 XU and ZHU: Role of Protein Kinases in Abscisic Acid and H2O2 Induced Antioxidant Defense in Maize摇 摇 摇
2. 2摇 Effects of pretreatment with ROS scavengers
on ABA鄄induced protein kinase activity increases
Reversible protein phosphorylation catalyzed by
protein kinases and phosphatases has been believed
to play central roles in ABA signal transduction. In
order to determine whether ABA鄄induced increases
in the activities of total protein kinases and calcium
dependent protein kinases resulted from the genera鄄
tion of ROS, ROS scavengers were used here. As
shown in Fig. 1C and 1D, pretreatment with ROS
scavengers such as Tiron (1,2鄄dihydroxybenzene鄄3,
5鄄disulphonic acid) and DMTU (Dimethylthiourea)
substantially reduced the ABA鄄induced activities of
total protein kinases and calcium dependent protein
kinases. These results suggested that ROS produc鄄
tions are required for ABA鄄induced the change of
protein kinase activities.
2. 3摇 Effects of pretreatment with protein kinase
inhibitors on ABA鄄induced protein kinase activi鄄
ty increases
In order to examine the biochemical characteriza鄄
tion of total protein kinase and calcium dependent pro鄄
tein kinase activities elevations induced by ABA, we
performed the standard solution in vitro kinase assay
(see “Experimental Procedures冶). As shown in Fig.
2A and 2B, the total protein kinase and calcium de鄄
pendent protein kinase activities induced by ABA were
almost inhibited in the presence of protein kinase in鄄
hibitor K252a compared with control level. Whereas,
the addition of H7 and KN鄄93 only partly blocked the
total protein kinase and calcium dependent protein ki鄄
nase activities induced by ABA. These results indica鄄
ted that increase of protein kinase activity induced by
ABA is sensitive to protein kinase inhibitor.
2. 4摇 Effects of pretreatment with protein kinase
inhibitors on activities of ABA鄄induced antioxi鄄
dant enzymes
To establish a link between protein phosphoryl鄄
ation and antioxidant defense induced by ABA, the
detached plants were pretreated with several protein
kinase inhibitors, such as k252a, a broad range pro鄄
tein kinase inhibitor (Kase et al., 1987), H7, a
protein kinase C inhibitor (Kumahara et al., 1999)
and KN鄄93, a CaMK inhibitor ( Lu and Feldman,
1997) respectively, and then exposed to ABA treat鄄
ment. As shown in Fig. 3, pretreatment with kinase
inhibitors significantly inhibited the increases in the
activities of SOD, CAT, APX and GR (Fig. 3) in鄄
duced by ABA. Furthermore, our data also proved
that antioxidant enzymes activities increases induced
by H2 O2 were blocked by protein kinases inhibitors
k252a, H7 and KN鄄93 (Xu, 2010). These results
suggested that protein phosphorylation is required for
ABA鄄induced antioxidant defense.
Fig. 2摇 Effects of pretreatment with protein kinase inhibitors on the total protein kinase activities and Ca2+ 鄄dependent protein kinase ac鄄
tivities in maize leaves treated by ABA. (A) Effects of protein kinase inhibitors on the total protein kinase activities. (B) Effects of pro鄄
tein kinase inhibitors on the Ca2+ dependent protein kinase activities. The values are the mean依SE (n=6) for three different experiments
082摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 植 物 分 类 与 资 源 学 报摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 第 33 卷
Fig.3摇 Effects of pretreatment with protein kinase inhibitors on the total activities of antioxidant enzymes in leaves of maize plants exposed to ABA
treatment. (A) SOD (superoxide dismutase). (B) CAT (catalase). (C) APX (ascorbate peroxidse) . (D) GR (glutathione reductase) .
The detached plants were pretreated with distilled water, DMSO, k252a, H7, Staurosporine for 4 h, and then exposed to 100 滋mol·L-1
ABA for 12 h. Detached plants treated with distilled water under the same conditions throughout the period of the experiment served as control.
The detached plants were treated as follows: 1, distilled water+distilled water (control); 2, DMSO+distilled water; 3, distilled water+
ABA; 4, 1 滋mol·L-1 k252a+ABA; 5, 10 滋mol·L-1 H7+ABA; 6, 1 滋mol·L-1 KN-93+ABA.
The values are the means依SE (n=6) of three different experiments. Means denoted by the same letter did not significantly differ at P<
0. 05 according to Duncan忆s multiple range test
2.5摇 ABA and H2O2stimulate a set of protein kinases
To investigate some potential kinases that are
involved in ABA鄄induced antioxidant defense, the
in鄄gel kinase assays with Histone III鄄S as a substrate
were applied on protein extracts from maize leaves
treated with ABA and H2O2 respectively. By analy鄄
zing protein solubilized samples, we identified a set
of protein kinases that were activated by ABA. Four
protein kinases with molecular masses of 66 kDa, 52
kDa, 49 kDa and 35 kDa were activated and dis鄄
played different kinetics respectively ( Fig. 4: A,
B). These bands were also observed after treatment
with H2O2, indicating that both ABA and H2O2 can
stimulate the four protein kinases (Fig. 4: C, D).
The activation of 52 kDa and 49 kDa protein kinases
occurred at 15 min after ABA treatment, peaked at
60 min and then high activity was maintained be鄄
tween 120 and 240 min after treatment. The 66 kDa
protein kinase activity peaked at 60 min and 35 kDa
protein kinase activity maximized at 30 min. Howev鄄
er, when treated with H2 O2, all the activation of
four protein kinases peaked at 30 min. These protein
kinases induced by ABA or H2 O2 are clearly time
course dependent ( Fig. 4: A, C). Further more,
the activation of four protein kinases also occurred in
a dose鄄dependent manner (Fig. 4: B, D).
1823 期摇 摇 XU and ZHU: Role of Protein Kinases in Abscisic Acid and H2O2 Induced Antioxidant Defense in Maize摇 摇 摇
Fig. 4摇 ABA and H2O2 activate a set of protein kinases in leaves of maize plants. (A) Time course for ABA鄄induced four protein kinases activa鄄
tion. (B) Dose dependence for ABA鄄induced four protein kinases activation. (C) Time course for H2O2 鄄induced four protein kinases activation.
(D) Dose dependence for H2O2 鄄induced four protein kinases activation. All experiments were repeated at least three times with similar results
2. 6摇 Fifty鄄two kDa protein is related to Ca2+鄄de鄄
pendent and would belong to CaMK family
Previous studies have shown that protein kina鄄
ses are involved in ABA signal pathway (Finkelstein
and Gibson, 2002; Fujii et al., 2007). To investi鄄
gate the kinase type activated by ABA in maize leav鄄
es, the modified in鄄gel kinase assays were per鄄
formed. When in the absence of free Ca2+ in the re鄄
action buffer, the 52 kDa kinase band was not detec鄄
ted, but the 49 kDa band was still observed (Fig.
5). These results suggested that the 52 kDa kinase
was a Ca2+ dependent protein kinase and could be
stimulated by ABA. The 49 kDa kinase is a Ca2+ in鄄
dependent protein kinase.
Fig. 5摇 ABA activates a 52 kDa Ca2+ 鄄dependent and CaM鄄stimulated
protein kinase. The sample from detached maize leaves treated with
100 滋mol·L-1 ABA were used for in鄄gel kinase, with 0. 25 mg·mL-1
histone III鄄S in the separating gel as a kinase substrate. Kinase activi鄄
ties were assayed in the presence of 1 mmol·L-1 Ca2+ or in the absence
of Ca2+ but containing 1 mmol·L-1 EGTA (left), and in the presence
of 1 mmol·L-1 Ca2+ and 14 滋g·ml-1 calmodulin ( right) . All experi鄄
ments were repeated at least three times with similar results
Previous work has shown that one calcium /
calmodulin鄄dependent kinase (CaMK) expression is
increased in response to ABA (Yang et al., 2004).
The 52 kDa kinase is shown to be induced by ABA
and possesses characteristic of Ca2+ dependent, sug鄄
gesting that it could be related to CaMKs. To test
this hypothesis, exogenous CaM was prepared and
added in the reaction buffer. The activity of 52 kDa
kinase in the presence of Ca2+ / CaM was much higher
than in the Ca2+ alone (Fig. 5). These results sug鄄
gested that the ABA鄄induced 52 kDa kinase might be鄄
long to CaMK family.
2.7摇 Four protein kinases activated by ABA and H2O2
participate in ABA鄄induced antioxidant defense
To further investigate whether the four protein
kinases activated by ABA participate in ABA鄄in鄄
duced antioxidant defense, the kinases inhibitors
previously mentioned were applied. ABA鄄induced
the four protein kinases activation was examined.
Pretreatment with k252a substantially blocked the
activation of the four protein kinases induced by
ABA compared with ABA treatment only. However
the activity of the 66 kDa protein kinase was com鄄
pletely inhibited particularly (Fig. 6). We also ana鄄
lyzed the effects of the protein kinase C inhibitors
H7 and CaMK specific inhibitor KN鄄93. The in鄄gel
autophosphorylation and histone鄄phosphorylating ac鄄
282摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 植 物 分 类 与 资 源 学 报摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 第 33 卷
tivity of the 66 kDa kinase induced by ABA could be
entirely inhibited by H7 and KN鄄93 (Fig. 6). Pre鄄
treatment with H7 and KN鄄93 substantially reduced
the ABA鄄stimulated 52 kDa protein kinase activity,
and partly blocked the activation of 49 kDa protein
kinase. The solvent dimethyl sulfoxide (DMSO) of
various inhibitors did not affect the four protein ki鄄
nase activity, compared with the control ( Fig. 6).
These results indicated that the four protein kinases
are involved in ABA鄄induced antioxidant defense.
Fig. 6摇 Effect of protein kinase inhibitors on ABA induced kinase ac鄄
tivation. Samples treated with 100 滋mol·L-1 ABA for various times,
were used for in鄄gel kinase assay, in the presence of 1 mmol·L-1
Ca2+ . The detached plants were treated as follows: 1, distilled water+
distilled water (control); 2, DMSO+distilled water; 3, distilled wa鄄
ter+ABA; 4, 1 滋mol·L-1 k252a+ABA; 5, 10 滋mol·L-1 H7+ABA;
6, 1 滋mol·L-1 KN-93+ABA. All experiments were repeated at least
three times with similar results
2. 8摇 Relationships between the four protein ki鄄
nases and ROS in ABA signaling
Previous studies (Pei et al., 2000; Jiang and
Zhang, 2001, 2002a) have shown that ABA can in鄄
duce the production of ROS such as H2O2, the ex鄄
pression of antioxidant genes encoding SOD, CAT,
and APX, and enhance the activities of these antiox鄄
idant enzymes in plant tissues. To further determine
the interrelationship among ABA, H2O2 and the four
protein kinases in ABA signaling, the ABA鄄deficient
maize vp5 mutant which interrupts ABA biosynthesis
early in the biosynthetic pathway was used (Guan et
al., 1998; Sharp et al., 2002). Treatment with 10
mmol·L-1 H2O2 resulted in a significant increase in
the activity of the 66 kDa and 49 kDa protein kinases
in the vp5 mutant type (Fig. 7: A). However only a
slight increase in the activity of the 35 kDa and 52
kDa protein kinases in the mutant was observed
(Fig. 7: A, B). These results indicated that the ac鄄
tivation of 66 kDa and 49 kDa is dependent on H2O2
or H2O2 accumulation and the 66 kDa and 49 kDa
protein kinases may act downstream of ROS in ABA鄄
induced antioxidant defense. In contrast, these re鄄
sults also suggest that 52 kDa and 35 kDa protein ki鄄
nase may act downstream ABA and upstream of ROS
in ABA鄄induced antioxidant defensive signaling.
Fig. 7摇 Effect of H2O2 on a set of protein kinases induced by ABA in
leaves of maize vp5 mutant. (A) Time course for H2O2 鄄induced four
protein kinases activation. ( B) Time course for H2 O treatment as
control for H2O2 鄄induced four protein kinases activation. All experi鄄
ments were repeated at least three times with similar results
3摇 Discussion
ABA regulates many important plant processes,
including seed germination, dormancy, seedling growth,
and stomatal aperture as well as in plants adapting to
their adverse environment ( Chow et al., 2004;
Yamaguchi et al., 2006). It has been well docu鄄
mented that ABA can cause the increased generation
of ROS, induce the expression of antioxidant genes,
and enhance the capacity of antioxidant defense sys鄄
tems in plants (Jiang and Zhang, 2004). ABA鄄in鄄
duced ROS production plays an important role in the
ABA signal transduction pathway, leading to the in鄄
duction of antioxidant defense systems (Hu et al.,
2005; Jiang and Zhang, 2002a, b, 2004). Howev鄄
er the detailed mechanisms about how ABA鄄induced
ROS production is transduced into the antioxidant
defense response remain elusive.
In the last decade, the mechanism of ABA鄄me鄄
3823 期摇 摇 XU and ZHU: Role of Protein Kinases in Abscisic Acid and H2O2 Induced Antioxidant Defense in Maize摇 摇 摇
diated signal transduction in ABA鄄induced antioxi鄄
dant defense has been investigated extensively. And
it has been documented that the MAPK cascade
plays an important role in ABA鄄induced antioxidant
defense and act downstream of ROS production in
leaves of maize plants (Zhang et al., 2006). How鄄
ever it remains unclear that whether other phospho鄄
rylation components participate in ABA鄄induced an鄄
tioxidant defense.
Protein phosphorylation is known to play a key
role in eukaryotic cell signaling and to be involved in
the regulation of many fundamental cellular events
(Shen et al., 2004). Previous work showed that re鄄
versible protein phosphorylation as an early and cen鄄
tral event in ABA signal transduction, at least in the
guard cell ( Schmidt et al., 1995; Leung et al.,
1997; Himmelbach et al., 2003; Sokolovski et al.,
2005). However, guard cells are highly specialized
cells, and it is not clear whether the model de鄄
scribed above is applicable to other ABA signal
pathway as well. In non鄄stomatal tissues, the cells
total protein phosphorylation levels in response to
ABA have not been identified yet.
Here our results showed that ABA and H2 O2
can activate transient increase in the activities of the
total protein kinases ( Fig. 1: A) and calcium de鄄
pendent protein kinases in maize leaves ( Fig. 1:
B). These data not only demonstrated that ABA and
H2O2 treatment stimulate activities of both the total
protein kinases and calcium dependent protein kina鄄
ses (Fig. 1: A, B), but also showed that H2 O2 is
required for ABA activating total protein kinases and
calcium dependent protein kinases (Fig. 1: C, D).
These results suggested that calcium dependent pro鄄
tein kinases and calcium independent protein kinases
might involve in other ABA signal transduction path鄄
way such as antioxidant defensive ubiquitously.
For years, it has been shown that ABA induces
ROS production, and ROS are required for the total
leaf activities of SOD, CAT, APX and GR increases
in maize ( Jiang and Zhang, 2002a, b), and that
treatment with ABA or H2O2 lead to a significant en鄄
hancement of activities of SOD, CAT, APX and GR
in leaves of maize seedlings, and these antioxidant
enzyme activities maximized at 12 h ( Jiang and
Zhang, 2002b). Our works not only demonstrated
that ABA or H2O2 induced increases in the activities
of antioxidant enzymes in leaves of maize seedlings
clearly (data not shown), but also showed that pro鄄
tein phosphorylation preceded antioxidant enzyme
activities obviously (Fig. 1).
In this study, we found that pretreatment with
protein kinase inhibitors reduced the antioxidant en鄄
zyme activity increases induced by ABA (Fig. 2),
so the antioxidant pathways in ABA signaling are
presumed to act through protein phosphorylation.
This is consistent with previous studies about MAPK
from maize plants (Zhang et al., 2006) and OXI1
from Arabidopsis (Rentel et al., 2004). Additional鄄
ly, our data also showed that pretreatment with
K252a, H7, KN鄄93 substantially blocked the activi鄄
ties of total antioxidant enzymes such as SOD, CAT,
APX and GR induced by ABA to distinct extent
(Fig. 2). These results proved that distinct protein
kinases might participate in ABA鄄induced antioxi鄄
dant defense through different pathways.
The fact that ABA treatment can enhance the
activities of total protein kinases and Ca2+ dependent
protein kinases indicated that there exists other pro鄄
tein components involving in ABA and ROS signa鄄
ling pathways (Fig. 4). In this report, according to
the results of the in鄄gel kinase assay, we identified a
class of ABA stimulated protein kinases from the
maize plants with molecular masses of 66 kDa, 52
kDa, 49 kDa and 35 kDa respectively (Fig. 4). Re鄄
markably, our results indicated that 52 kDa protein
kinase has the typical features of CaMK because of
its calmodulin dependent activation (Fig. 5). Addi鄄
tionally, the activity of the 52 kDa protein kinase
was sensitive to KN鄄93, which was used as a CaMK
II inhibitor (Fig. 6). Furthermore, the 52 kDa pro鄄
tein kinase activity which has characteristic of Mn2+
activation can be blocked by exogenous or endoge鄄
nous KN鄄93 or W鄄7 (data unpublished). These re鄄
482摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 植 物 分 类 与 资 源 学 报摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 第 33 卷
sults strongly suggested that the 52 kDa protein ki鄄
nase is a Ca2+ / calmodulin鄄dependent and binding
protein kinase.
In the present study, using the ABA鄄deficient
maize vp5 mutant, these data revealed that 66 kDa
and 49 kDa protein kinases might act downstream of
reactive oxygen species in ABA鄄induced antioxidant
defense, and that 52 kDa and 35 kDa protein kinase
may act downstream ABA and upstream of reactive
oxygen species in ABA鄄induced antioxidant defen鄄
sive signaling. These results, taken together, indi鄄
cated that at least four protein kinases may play cru鄄
cial roles in ABA induced antioxidant defensive sig鄄
naling pathways. However, for now, the identities
and detailed characteristic properties of these kinases
remain unknown. Therefore, molecular properties
and functional roles of these protein kinases in ABA
and H2O2 鄄induced antioxidant defensive signaling are
being investigated in our laboratory.
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