全 文 ：植物学通报Chinese Bulletin of Botany 2007, 24 (4): 444-451, www.chinbullbotany.com
收稿日期: 2007-04-28; 接受日期: 2007-05-30
基金项目: 国家自然科学基金(No. 30470895)、河南省高校新世纪优秀人才支持计划(No. 2005HANCET-06)和河南省青年骨干教师资
助计划
* 通讯作者。E-mail: songcp@henu.edu.cn
.实验简报.
SB202190调节蚕豆保卫细胞中 SA诱导 H2O2产生
江静, 韩栓, 宋纯鹏 *
河南大学植物逆境生物学重点实验室和生命科学学院, 开封 475001
摘要 运用激光共聚焦扫描技术, 在p38 MAP激酶专一抑制剂SB202190处理下, 探索植物促分裂原活化蛋白激酶(mitogen-
activated protein kinase, MAP激酶)介导蚕豆(Vicia faba)保卫细胞中H2O2为代表的活性氧(reactive oxygen species, ROS)
信号机制, 发现: p38 MAP激酶专一抑制剂SB202190处理没有导致蚕豆保卫细胞中H2O2和Ca2+探针荧光强度增强, 与水杨
酸 (salicylic acid, SA) 或脱落酸 (abscisic acid, ABA) 迅速加强2种探针荧光强度形成鲜明对比; 而该抑制剂分别与SA和ABA
共同处理, 前者H2O2探针荧光强度没有增加, 而后者荧光强度仍然能够增加; 而进一步使用Ca2+螯合剂BAPTA和SB202190
＋SA共同处理, H2O2探针荧光强度没有增加。这些结果初步表明: 无论胞质Ca2+浓度高低, SB202190调节蚕豆保卫细胞中
SA诱导H2O2产生, 但是不调节植物逆境信使分子ABA 此类的反应。因此推测, 植物细胞中可能有类似动物和酵母细胞中的p38
MAP激酶类, 并可能专一调节植物保卫细胞中H2O2信号通路。据我们所知, 这是首次报道SB202190和SA共同调节植物保卫
细胞中ROS信号过程。
关键词 ABA, H2O2, p38 MAP 激酶, SA, SB202190, 保卫细胞
江静, 韩栓, 宋纯鹏 (2007). SB202190调节蚕豆保卫细胞中 SA诱导 H2O2产生. 植物学通报 24, 444-451.
已有研究证明活性氧(reactive oxygen species,
ROS)是植物应答胁迫刺激信号过程中的第二信使, 其机
制倍受关注(Neill et al., 2002; Desikan et al., 2004)。
植物激素脱落酸(abscisic acid, ABA)依赖NADPH氧化
酶诱导 ROS产生、胞质 Ca2+浓度升高以及蛋白磷酸
化 / 去磷酸化等过程应答胁迫(Nei l l e t a l . , 2002;
Desikan et al., 2004)。而植物激素水杨酸(salicylic
acid, SA)则通过提高超氧化物歧化酶(CuZn-superoxide
dismutase, SOD)同时降低过氧化氢酶(catalase, CAT)
活性、并和 H2O2相互促进、形成信号自我放大反馈
环(self-amplifying feedback loop), 放大氧化信号, 提
高植物抗胁迫能力(Shirasu et al., 1997; Mori et al.,
2001; Kawano, 2003; Suhita et al., 2004)。研究证
明蚕豆(Vicia faba)保卫细胞中, 依赖NADPH氧化酶活
性的超氧阴离子(O2-. ) 产生反应在SOD催化O2-. 歧化为
H2O2的反应之后, 同时, SA诱导的O2-. 浓度升高反应
在胞内 Ca2+浓度升高之前(Mori et al., 2001)。可见,
认识保卫细胞ROS产生和代谢反应机制专一性还需要
更深入的研究。
酵母和动物细胞中MAP激酶类包括三亚族: ERK
类、p38 MAP激酶类和 JNK类, 特点是: 序列高度保
守、成员众多、存在广泛, 经过“M A P K K 激酶、
MAPK激酶和MAP激酶”三级磷酸化(cascade)形式
应答多种刺激信号(Morris, 2001), 其中一些成员与ROS
共同调节植物细胞各种胁迫信号转导受到广泛关注
(Pitzschke and Hirt, 2006; Zhang et al., 2006)。p38
MAP激酶最初是在酵母细胞应答环境渗透刺激反应中
被发现的, 以 STE11 / SSK2 (MAPKK激酶)、PBS2
(MAPK激酶)和 p38/HOG1 (high osmotic glycerol 1)
(MAP激酶)级联 (cascade) 途径调节胞内渗透物质浓度,
445江静等: SB202190调节蚕豆保卫细胞中SA诱导H2O2产生
最后被蛋白酪氨酸磷酸酶 ( p r o t e i n t y r o s i n e
phosphatase, PTP) 2/3 去磷酸化而灭活(Morris,
2001)。令人感兴趣的是, p38 MAP激酶和 H2O2共同
调节酵母和动物细胞中各种胁迫信号过程 (Czubryt et
al., 2000; Niwa et al., 2001; Kuruganti et al., 2002;
Sheikn-Hamad and Gustin, 2004; Kim and Lee 2005;
K i m e t a l . , 2 00 6) , 而 NAD PH 氧化酶和 Ca 2+
(Kreideweiss et al., 1999; Ramachandiran et al., 2002;
Hu et al., 2005; Rodríguez-Gabriel and Russell, 2005;
Hsieh and Papaconstantinou, 2006)是其中关键成分。
虽然一些研究结果表明植物细胞中可能存在类似的p38
MAP激酶通路信号成分, 比如植物细胞中存在酵母p38
MAP激酶的同源基因 (Pöpping et al., 1996; Urao et
al., 1999) 及其激酶活性 (Li et al., 1998; Munnik et
al., 1999; Hoyos and Zhang, 2000; Komis et al., 2004)
, 但是迄今未见植物细胞中有关这些激酶活性与ROS关
系的报道。
已有研究发现, MEK1/2 调节ABA诱导的H2O2产
生和代谢, 而不调节SA诱导的ROS代谢反应(Grant et
al., 2000; Yuasa et al., 2001; Jiang et al., 2003;
Ichimura et al., 2006; Zhang et al., 2006), 而SA激活
的 MAP激酶却对 ABA不敏感(Hoyos and Zhang,
2000)。据此推测 SA和 ABA诱导的 ROS信号转导通
路可能被不同MAP激酶成员调节, 但其机制尚需探索。
本文研究p38 MAP激酶专一性抑制剂SB202190调节
SA和ABA诱导H2O2产生机制发现, 无论Ca2+浓度高
低, SB202190可以调节蚕豆保卫细胞中SA诱导H2O2
产生过程, 却没有调节 ABA类似反应。
1 材料与方法
1.1 实验材料
蚕豆(Vicia faba)种子用 75%乙醇消毒, 在生化培养箱
中催芽, 待胚根长约0.5 cm时, 播种于培养土 (培养土:
蛭石＝ 2:1)中。每天光照 10小时, 光照强度为 0.20-
0.30 mmol.m-2.s-1, 昼夜温度分别为(25 ± 2)°C和(19
± 2)°C。
1.2 实验方法
1.2.1 试剂配制
H2DCFDA、SB202190和Fluo-3 AM分别溶解于dim-
ethyl sulfoxide (DMSO), 制成母液, 避光操作, 分装,
-30 °C冷冻保存。
SB202190为Calbiochem (La Jolla, CA, USA)公
司产品, ABA、SA、MES、Fluo-3 AM、BAPTA、
Eserine及DMSO均为 Sigma (St. Louis,USA)公司
产品。H 2DCFDA 为 Molecular Probe (Eugene
Oregon, USA)公司产品。其余试剂均为国产分析纯
级别的产品。
1.2.2 荧光探针孵育
H2O2探针的导入: 将新鲜表皮条放入H2O2探针导入缓
冲液(50 mmol.L-1 KCl + 10 mmol.L-1 Tris, pH7.18),
加入H2DCF-DA母液, 其最终浓度为50 mmol.L-1, 混合
均匀, 25°C, 避光孵育10-15分钟。H2DCF-DA容易进
入细胞, 胞内 H2O2将其氧化为具有荧光活性的化合物
DCF, 适合于激光共聚焦扫描 (laser scanning confocal
microscope, LSCM) 分析。
Ca2+探针的导入: 将新鲜的表皮条放入Ca2+探针导
入缓冲液(50 mmol.L-1 KCl + 10 mmol.L-1 Tris + 5
mmol.L-1 CaCl2 + 0.5 mmol.L-1 Eserine, pH7.18), 加
入Fluo-3 AM母液, 使其最终浓度达到30 mmol.L-1, 混
合均匀, 30°C, 避光孵育 30-45分钟。
1.2.3 激光共聚焦扫描及数据分析方法
参照Zhang等(2001)的方法, 稍加改动。取出已导入探
针的表皮条, 洗去表面多余探针, 然后将其固定在合适
的小培养皿底部, 加入适量缓冲液, 放在显微镜(Bio-Rad
MicroRadiance)下。根据需要分别加入 ABA、SA、
SB202190和BAPTA等试剂刺激保卫细胞。以Time-
course软件采集数据。为保证数据可比性, 所有实验
中的LSCM工作条件手工设定为:Ex=488 nm, Em=525
± 15 nm, Power=10%, Zoom=4, Frame=512×512, 中
速扫描。
记录结果分析: 图片采用photoshop软件处理。随
446 植物学通报 24(4) 2007
机选出5个Time-course记录的保卫细胞探针荧光强度
数据分析。
2 结果与分析
2.1 SB202190抑制保卫细胞中SA诱导H2O2产生
植物气孔保卫细胞是研究逆境信号转导模式系统。P38
MAP激酶广泛参与动物细胞 H2O2信号过程 (Sheikh-
Hamad and Gustin, 2004)。气孔保卫细胞中 H2O2
和 SA相互作用, 放大胁迫信号(Mori et al., 2001;
Suhita et al., 2004)。为了探索 p38 MAP激酶类是
否参与SA诱导H2O2产生过程, 我们用特异性抑制剂
SB202190处理, 观察SA诱导H2O2产生的情况变化。
对照处理 , 缓冲液、S B 2 0 2 1 9 0 及其类似物
SB202474都不能促进H2O2荧光探针DCF荧光强度升
高(图 1A1-A3, D)。在缓冲液中单独加 SA, 保卫细胞
DCF 荧光强度迅速增强(图 1B1-B3)。但是, SA 和
SB202190共同处理, 保卫细胞DCF荧光强度却没有上
升 (图 1C1-C3), 图 1D显示了其对比值。这些结果表
明 SB202190阻断了 SA诱导 H2O2的产生。
2.2 SB202190不能抑制保卫细胞中ABA诱导
H2O2产生
H2O2是植物细胞中胁迫反应的第二信使 (Neill et al.,
2002; Desikan et al., 2004)。为了探测 SB202190
在不同上游信号分子诱导产生H2O2过程中的作用, 我
们继续观察此抑制剂是否影响胁迫信使 A B A 诱导
H2O2产生。
无ABA时, H2O2荧光探针H2DCF-DA荧光强度不
升高(图 2); 而用 ABA处理时其荧光强度迅速增强(图
2)。与 SA处理情形 (图 1)类似。但是 , SB202190
存在时 , ABA 诱导 DCF 荧光强度仍然增强 (图 2) ,
与ABA单独处理DCF荧光强度增强的结果一样, 说
明 SB202190 不影响 ABA 诱导 H 2O 2的产生。这
与该抑制剂阻断 SA诱导H 2O 2产生结果(图 1)形成
鲜明对比。
2.3 SB202190不依赖胞质Ca2+抑制SA诱导H2O2
产生
Ca2+不仅可以和 p38 MAP激酶共同调控动物细胞中
H2O2信号转导(Blanc et al., 2004), 而且是植物细胞H2O2
产生过程的重要影响因子(Pei et al., 2000; Mori et al.,
2001; Jiang and Zhang, 2003)。所以有必要调查Ca2+
在 SB202190阻断 SA诱导产生H2O2过程中的功能。
如表1所示, 单独的SB202190处理和空白对照处
理一样, 保卫细胞内Ca2+探针 Fluo-3荧光强度均未升
高, 这表明抑制剂本身并不影响Ca2+浓度变化; SA能
使Fluo-3荧光强度增加, 验证Ca2+参与保卫细胞SA
信号过程; 而SA+SB202190处理不影响Fluo-3荧光
强度增加(表1), 说明Ca2+存在情况下, SB202190能
够发挥作用。
Ca2+络合剂 BAPTA处理不影响 DCF荧光强度
(表 1)。使用 BAPTA和 SA+SB202190联合处理孵育
了H2O2探针H2DCF-DA的表皮条, 扫描过程中没有发
现保卫细胞DCF荧光强度变化(表1)。此结果表明Ca2+
不存在情况下, SB202190依然调节保卫细胞中 SA诱
导产生 H2O2过程。
3 讨论
ROS产生和代谢是植物应答逆境反应的关键步骤, 并与
Ca2+信使、MAP激酶交叉调节 ABA和 SA刺激的生
表 1 不同处理下 2种探针荧光强度
Table 1 The fluorescent intensity of two probes by various
treatments
Treatments DCF Fluo-3
SA 112.3±20.8 101.94±21.36
SB202190 20.28±17 32.36±22.32
SA+SB202190 33.68±18 103.77±19.33
SA+SB202474 118.3±19.32 102.1±17.87
BAPTA 32.07±19.15
SA+SB202190+BAPTA 19.76±15.19
表中荧光强度数据是实施处理 10分钟时的 LSCM记录数据
DCF and Fluo-3 fluorescent intensity are monitored by the vari-
ous treatments for 10 min. Results are the averages ± SE from
at least 3 independent experiments
447江静等: SB202190调节蚕豆保卫细胞中SA诱导H2O2产生
理生化过程(Mori et al., 2001; Suhita et al., 2004)。
虽然动物细胞中p38 MAP激酶被证明广泛调节ROS信
号过程, 但植物细胞中是否存在这样的机制尚未见报
道。本研究发现 , p 3 8 M A P 激酶专一性抑制剂
图 1 SB202190 阻断蚕豆保卫细胞 SA诱导 H2O2产生
(A1-A3) 空白处理时保卫细胞中 ROS探针荧光变化记录, (A0)为相应的透射光图;
(B1-B3) 100 mmol.L-1 SA处理时 ROS产生情况记录, (B0)为相应的透射光图;
(C1-C3) 100 mmol.L-1 SA+10 mmol.L-1 SB202190处理下 ROS产生情况, (C0)为相应的透射光图;
(D) 不同处理统计结果。
表皮条事先孵育H2O2探针H2DCFDA
Figure 1 SB202190 block SA-induced H2O2 generation in Vicia guard cells
(A1-A3) no change in ROS generation in guard cell by blank treatment, (A0) from the bright-light image;
(B1-B3) ROS elevation by 100 mmol.L-1 SA, (B0) the corresponding bright-light image;
(C1-C3) the change in ROS generation by 100 mmol.L-1 SA+ 10 mmol.L-1 SB202190 treatment, (C0) the corresponding bright-light image;
(D) the measurements of DCF fluorescent intensity by the various treatments. Results are the averages± SE from at least 3
independent experiments.
Guard cells loaded with H2DCFDA
448 植物学通报 24(4) 2007
SB202190阻断 SA诱导 H2O2浓度升高, 而且Ca2+螯
合剂BAPTA没有阻断SB202190的这种作用。这些结
果初步揭示, 植物细胞中存在类似动物细胞中p38 MAP
激酶调节H2O2信号传递的反应机制。但该抑制剂不影响
ABA启动的这些反应, 暗示了H2O2信号途径的专一性。
生理生化实验表明, SB202190可以结合活性形式
和非活性形式的p38 MAP激酶, 进而非常专一地阻断它
的下游作用机制(Frantz et al., 1998; Nemoto et al.,
1998; Lee et al., 1999), 因此被用来研究p38 MAP激
酶和ROS调节动物细胞中各种各样的胁迫机制, Ca2+参
与此过程的调节(Manthey et al., 1998; Montero et al.,
2002; Cicconi et al., 2003; Prakash et al., 2005)。
与动物细胞中反应类似, SA通过调节植物细胞中
ROS产生和代谢来帮助植物应答环境胁迫, 提高生存能
力(Mori, 2001)。SA诱导 H2O2产生和积累机理是: 一
方面SA可以提高植物体SOD酶活性, 加速O2-. 歧化为
H2O2 (Rao et al., 1997), 另一方面 SA与一些 CAT类
蛋白 -水杨酸结合蛋白(salicylic acid binding protein,
SABP) 结合而抑制CAT降解H2O2的能力 (Slaymaker
et al., 2002)。已证明气孔保卫细胞中 H2O2和 SA相
互作用, 放大胁迫信号(Mori et al., 2001; Suhita et al.,
2004), 因此, SB202190阻断SA诱导ROS产生(图1),
暗示蚕豆保卫细胞中类似 p38MAP激酶活性可能正调
节SOD活性升高, 或者同时正调节SA与CAT结合, 加
速 H2O2产生和积累, 放大胁迫信号。
Ca2+不仅可以和p38 MAP激酶共同调控动物细胞
中H2O2信号转导(Blanc et al., 2004), 而且是植物细胞
H2O2产生过程的重要影响因子(Pei et al., 2000; Mori
et al., 2001; Jiang and Zhang, 2003)。与HeLa细胞
研究结果(Montero et al., 2002)相似, 单独的SB202190
不影响蚕豆保卫细胞胞质Ca2+浓度(表1)。所以, SA和
SB202190联合处理, 胞质Ca2+探针荧光强度仍然升高
(表 1)。虽然研究显示 SA诱导 SOD活性升高, 并依赖
Ca2+加速O2-. 歧化为H2O2 (Rao et al., 1997; Slaymaker
et al., 2002; Kawano, 2003), 但平行证据表明, 蚕豆
保卫细胞中NADPH氧化酶催化O2-. 产生的反应发生在
SOD催化O2-. 为 H2O2反应之后, 同时, SA诱导O2-. 浓
度升高反应在胞内 Ca2+ 浓度升高之前(Mori et al.,
2001)。综合这些结果, Ca2+ 浓度升高与 H2O2产生和
积累反应可能是平行进行的。所以, 使用 BAPTA后,
SB202190对SA诱导H2O2产生阻断作用没有被解除, 或
者说 Ca2+ 浓度升高和SB202190阻断SA诱导H2O2产生
共同出现的结果(表 1)不难理解。可以想象, SB202190
专一作用于SA诱导H2O2产生信号路段, 而没有触及Ca2+
信号通路。这些结果很好地回应了Li等(1998)的报道: 无
论Ca2+还是EGTA处理条件下, 蚕豆保卫细胞都有高活
性的38 kDa蛋白激酶出现。因此推测, 植物细胞中p38
MAP激酶活性不仅调节氧化胁迫信号转导, 而且可能有
依赖 Ca2+ 和不依赖 Ca2+两种方式并存。
本研究结果显示, SB202190 没有调节 ABA诱导
H2O2信号机制(图2), 佐证H2O2作为信号分子的特性。
图 2 SB202190 不能调节蚕豆保卫细胞中 ABA诱导 H2O2产生
ABA和 ABA+SB202190处理时, 保卫细胞内 DCF荧光强度统计
结果。数据是 4 个以上不同细胞内荧光强度平均值
Figure 2 SB202190 do not block ABA-induced H2O2 genera-
tion in Vicia guard cells
The measurements of DCF fluorescent intensity are at the pres-
ence of ABA or ABA+SB202190 for before treatment, 5 min,
and 10 min. Results are the averages ± SE from at least 4
independent experiments
449江静等: SB202190调节蚕豆保卫细胞中SA诱导H2O2产生
已有研究表明, ABA可以依赖、也可以不依赖 Ca2+ 调
节ROS产生 (Fedoroff, 2006), 并可能由其它MAP激
酶家族成员专一调节, 比如, MEK1/2专一调节ABA诱
导 ROS产生 (Grant et al., 2000; Jiang and Zhang,
2003; Jiang et al., 2003; Fedoroff, 2006), 而不调节
SA诱导 H2O2产生(Grant et al., 2000; Jiang et al.,
2003)。Li等(1998) 报道蚕豆保卫细胞中38 kDa和57
kDa蛋白激酶同时被磷酸化激活, 暗示除38 kDa蛋白激
酶外, 还有其它MAP激酶类参与保卫细胞ABA信号转
导。2C型蛋白磷酸酶 ABI1和 ABI2调节 ABA及其下
游 H2O2信号通路 (Murata et al., 2001; Meskiene et
al., 2003), 进一步说明不同MAP激酶成员分享ROS信
号通路, 因为 p38MAP激酶是被 PTP2/3, 而不是被
PP2C类去磷酸化。ABA和 SA拉动的 ROS产生和代
谢机制既相似, 又各具特色, 胞质Ca2+和蛋白激酶 / 磷
酸酶顺应特色而发挥不同的调节作用。
由此可见, 作为信号分子的H2O2, 它的产生和灭活
都有特异性, Ca2+和p38 MAP激酶参与SA诱导H2O2
的产生, 显示H2O2信号途径的复杂性和专一性。MAP
激酶家族成员可能是实现ROS产生和代谢信号途径特
性的基础。
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SB202190 Modulate Salicylic Acid-induced H2O2 Generation
in Vicia Guard Cells
Jing Jiang, Shuan Han, Chunpeng Song*
Laboratory of Plant Stress Biology and Colege of Life Sciences, Henan University, Kaifeng 475001, China
Abstract Using laser scanning confocal microscopy, we tested the mechanism of mitogen-activated protein kinase (MAPK)
regulation of salicylic acid (SA)- or abscisic acid (ABA)-induced reactive oxygen species H2O2 elevation in Vicia guard cells.
SB202190, a special inhibitor of p38 MAPK, blocked the elevation of H2O2 by SA but not ABA, whereas SB202190 alone could not
increase H2O2 or Ca2+ level in guard cells. The combination of BAPTA, the chelator of Ca2+, and SA+SB202190 could not increase
H2O2 level, which suggests that activation of p38 MAPK-like especially modulates H2O2 signaling in plant cells, regardless of Ca2+
level, similar to the mechanism in mammalian or yeast cells. To our knowledge, this is the first report that SB202190 regulates SA-
induced ROS signaling in plant cells.
Key words ABA, H2O2, p38 MAP kinase, SA, SB202190, Vicia guard cells
Jiang J, Han S, Song CP (2007). SB202190 modulate salicylic acid-induced H2O2 generation in Vicia guard cells. Chin Bull Bot 24,
444-451.
* Author for correspondence. E-mail: songcp@henu.edu.cn
(责任编辑: 韩亚琴)