南京农业大学学报  2018, Vol. 41 Issue (2): 321-329   PDF    
http://dx.doi.org/10.7685/jnau.201704018
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文章信息

宋文敏, 孙嫚嫚, 蒋明义
SONG Wenmin, SUN Manman, JIANG Mingyi
水稻中SAPK8/9/10与OsRbohB/E蛋白互作在ABA诱导H2O2产生中的作用
Functional analysis of the interaction between SAPK and OsRboh in abscisic acid-induced H2O2 production in rice
南京农业大学学报, 2018, 41(2): 321-329
Journal of Nanjing Agricultural University, 2018, 41(2): 321-329.
http://dx.doi.org/10.7685/jnau.201704018

文章历史

收稿日期: 2017-04-12
水稻中SAPK8/9/10与OsRbohB/E蛋白互作在ABA诱导H2O2产生中的作用
宋文敏 , 孙嫚嫚 , 蒋明义     
南京农业大学生命科学学院, 江苏 南京 210095
摘要[目的]本文旨在验证水稻中SAPK8/9/10与OsRbohB/E蛋白的互作及其在ABA诱导的H2O2产生中的作用。[方法]先采用酵母双杂交系统进行初步的SAPK8/9/10与OsRbohB/E蛋白的互作分析,然后采用双分子荧光互补(BiFC)、GST-pull down和体外磷酸化技术验证其互作。为了进一步探讨SAPKs与OsRbohs在ABA信号转导中的作用,采用水稻原生质体瞬时体系分析ABA处理时SAPKs与OsRbohs对H2O2产生的影响。[结果]SAPK8/9/10与OsRbohB和OsRbohE在体内、外存在相互作用,且OsRbohs是SAPKs磷酸化的底物。与对照组相比,水稻原生质体瞬时过表达SAPK9/10SAPKs-OE)和OsRbohB/EOsRbohs-OE)组中H2O2的产生明显增加,且ABA处理后增加趋势增强;瞬时沉默SAPKs(ds-SAPKs)和OsRbohs(ds-OsRbohs)组中H2O2的产生明显下调,且ABA诱导的H2O2增加在这些原生质体中也均被抑制;SAPKs-OE+ds-OsRbohs、ds-SAPKs+OsRbohs-OE组中H2O2的产生表现出不同程度的减少。[结论]SAPKs与OsRbohs共同参与调节ABA诱导的H2O2的产生。
关键词脱落酸   应激激活蛋白激酶   OsRboh   蛋白互作   过氧化氢   
Functional analysis of the interaction between SAPK and OsRboh in abscisic acid-induced H2O2 production in rice
SONG Wenmin, SUN Manman, JIANG Mingyi    
College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China
Abstract: [Objectives] The aim of this study is to identify the interaction between rice sucrose nonfermenting1-related protein kinase 2 SAPKs and rice NADPH oxidases OsRbohs, and its role in ABA-induced H2O2 production in rice. [Methods] To clarify the interactions between SAPK8/9/10 and OsRbohB or OsRbohE, a yeast two-hybrid system was firstly used, and then glutathione S-transferase(GST) pull-down assay, bimolecular fluorescence complementation(BiFC) analysis and in vitro phosphorylation test were performed to confirm the interactions. To investigate the roles of SAPKs and OsRbohs in ABA-induced H2O2 production, a transient gene expression analysis and a transient RNA interference(RNAi) test in rice protoplasts were also used. [Results] Our results showed that OsRbohB/E interacted with SAKP8/9/10, and they were phosphorylated by SAPK8/9/10, indicating that OsRbohB/E were phosphorylation substrates of SAPK8/9/10. Further, under the control conditions, the transient over-expression of SAPK9/10 or OsRbohB/E increased the production of H2O2 in the protoplasts, and the RNAi silencing of SAPK9/10 or OsRbohB/E decreased the production of H2O2. ABA treatment induced a significant increase in the production of H2O2 in the control protoplasts, and the increases were further enhanced in the protoplasts transiently expressing SAPK9/10 or OsRbohB/E, but were inhibited in the protoplasts transiently silenced SAPK9/10 or OsRbohB/E. Moreover, the transient expression analysis in combination with the transient RNAi test in protoplasts also showed that both SAPKs and OsRbohs were required for ABA-induced H2O2 production. [Conclusions] These results indicate that both SAPKs and OsRbohs coordinately regulate the production of H2O2 in ABA signaling.
Key words: abscisic acid (ABA)    stress-activated protein kinase (SAPK)    OsRboh    protein-protein interaction    hydrogen peroxide   

脱落酸(abscisic acid, ABA)是植物体内一种重要的激素分子, 在调控植物生长发育和适应各种环境胁迫的过程中起着重要作用。植物响应干旱胁迫的一条有效途径是胞内ABA迅速积累, 继而启动一系列响应胁迫的反应, 包括诱导气孔关闭、积累渗透调节物质和增强抗氧化防护系统等[1-4]。ABA信号转导途径是一个非常复杂、多种细胞组分参与ABA信号转导的调控过程。之前的报道指出, 活性氧(reactive oxygen species, ROS)作为第二信使参与干旱胁迫下ABA调控的适应性生理反应[5]

植物细胞代谢过程中产生的ROS主要包括超氧阴离子()、单线态氧(1O2)和羟基自由基(HO·)以及过氧化氢(H2O2)等。NADPH氧化酶已经被证实是植物细胞ROS的重要来源[2, 6]。水分胁迫下植物体内ABA的积累能诱导编码NADPH氧化酶的基因表达并且增加其活性, 产生较多的ROS, 从而激活ABA信号级联反应, 增强植物对水分胁迫的耐性[7-8]。拟南芥基因组中有10个编码NADPH氧化酶催化亚基的基因(AtrbohA~J)[8], 而水稻基因组中则有9个基因(OsrbohA~I)[9]。在ABA诱导的拟南芥保卫细胞气孔关闭过程中, AtrbohDAtrbohF是ROS产生所必需的[8]。在水稻中, ABA处理诱导OsRbohBOsRbohE表达上调, 暗示这2个基因可能参与ABA诱导的H2O2产生[10-11]

蔗糖非酵解型蛋白激酶(sucrose non-fermenting 1-related protein kinase, SnRK)是广泛存在于植物中的一类Ser/Thr类蛋白激酶, 在植物抵抗逆境胁迫过程中发挥了重要作用[12-13]。依据基因结构及蛋白质序列同源性进行分析, 植物SnRK相关蛋白激酶基因家族可以分为SnRK1、SnRK2和SnRK3共3个家族, 其中SnRK2家族主要在植物响应ABA信号和抗逆信号转导中发挥重要的调节作用[14]。拟南芥中SnRK2家族的OST1(SnRK2.6)已被证实作用于产生ROS的上游[15]。OST1已经被显示能够直接与NADPH氧化酶中的AtrbohF互作, 磷酸化AtrbohF的Ser13和Ser174[16], 然而这种磷酸化的生理重要性还没有被证实。水稻基因组中包含10个SnRK2成员, 依次被命名为SAPK 1~SAPK10, 但只有与OST1同源的SAPK8、SAPK9和SAPK10激酶活性可被ABA诱导上调[17]。然而, 水稻中这些SnRK2激酶与Osrbohs的关系及其在ABA诱导的H2O2产生中的作用尚有待证实。因此, 本研究利用酵母双杂交、双分子荧光互补(bimolecular fluorescence complementation, BiFC)、谷胱苷肽巯基转移酶下拉试验(glutathione S-transferase pull down, GST-pull down)以及体外磷酸化技术, 分析了水稻中SAPK8/9/10与OsRbohB和OsRbohE在体内、外的相互作用关系; 同时通过水稻原生质体瞬时表达体系研究了这些SAPK与OsRboh在ABA诱导H2O2产生中的作用。

1 材料与方法 1.1 试验材料

本研究采用粳型常规水稻(Oryza sativa)品种‘徐稻4号’为试验材料。

1.2 试验方法 1.2.1 酵母双杂交试验

根据SAPK 8/9/10基因序列与pGDKT7图谱以NdeⅠ和EcoRⅠ酶切位点构建SAPK 8/9/10 -pGBKT7诱饵载体, 根据OsRbohB/E基因序列与pGADT7图谱构建OsRbohB/E-pGADT7靶蛋白载体。用ProQuest酵母双杂交系统(Invitrogen)分别转化酵母Y2和Y187细胞, 在SD-Leu/-Trp或者SD-Trp/-Leu/-His上融合培养。以加入X-α-gal的SD-Trp培养基培养pGBKT7空载、SAPK8/9/10-pG-BKT7单菌落的涂布, 以验证SAPK8-10自激活作用。

1.2.2 双分子荧光互补(BiFC)试验

根据SAPK 8/9/10OsRbohB/E序列以及pSPYNE、pSPYCE质粒载体图谱, 构建SAPK8/9/10-YFPN与OsRbohB-YFPC组合, SAPK8/9/10-YFPC与OsRbohE-YFPN组合的质粒载体组合, 并设置对照组, 利用基因枪轰击洋葱表皮, 25 ℃培养16 h后于激光共聚焦显微镜下观察黄色荧光[18]

1.2.3 谷胱甘肽转移酶下拉试验(GST-pull down)

使用Primer Premier 5.0软件设计SAPK 8/9/10上游带EcoRⅠ酶切位点和下游带XhoⅠ酶切位点的引物, 以SAPK8/9/10-T载体测序正确无突变的质粒为模板, 将SAPK8/9/10全长序列构建到带有GST标签的原核表达载体pGEX-4T-1上; 设计带有BamHⅠ和EcoRⅠ酶切位点的OsRbohB N端(355个氨基酸)的上、下游引物, 克隆OsRbohB N端片段, 并将其连接到pET30a-c载体上, 构建His-OsRbohB原核表达载体。采用MagneGSTTM Pull-Down System试剂盒(Promega)检测SAPK8/9/10与OsRbohB/E的体外互作。将转化有GST空载和SAPK8/9/10-GST菌裂解后与预洗过的MagneGSTTM磁珠结合, 加入目的蛋白OsRbohB-His或OsRbohE-His以及反应缓冲液, 混匀后在4 ℃旋转仪上反应2 h, 然后用MagneGSTTM Binding/Wash Buffer洗涤磁珠, 去除非特异性结合的杂蛋白, 洗涤3次后加入适量蛋白上样缓冲液, 煮样, SDS-PAGE电泳, 并用His一抗进行Western Blotting检测。

1.2.4 体外磷酸化凝胶激酶分析

参照Zhang等[19]的方法并稍加修改。向GST-SAPK8/9/10与His-OsRbohB/E纯化后的蛋白样品中加入激酶反应液(25 mmol·L-1 Tris、pH7.5, 12 mmol·L-1 MgCl2, 1 mmol·L-1 DTT, 0.1 mmol·L-1 Na3VO4, 5 mmol·L-1 CaCl2, 0.5 mg·mL-1 MBP, 200 mmol·L-1 ATP, 50 μCi γ32-P-ATP), 总体积不超过40 μL, 30 ℃下反应30 min, 加入8 μL 5×SDS上样缓冲液, 煮沸5 min。SDS-PAGE电泳后将凝胶转入5%(体积分数)三氯乙酸(TCA)和1%(体积分数)焦磷酸钠(NaPPi)混合液中清洗并终止反应, 清洗3次, 每次5 min, 以彻底除去游离的γ32-P-ATP。用玻璃纸将凝胶制成干胶, 用Typhoon打磷屏显影。

1.2.5 双链RNA(double-stranded RNA, dsRNA)的体外合成与纯化

使用RiboMAXTM Large Scale RNA Production System-T7试剂盒(Promege), 并按照说明书进行体外转录dsRNA, 以酚/氯仿抽提并纯化, 用RNA-free水溶解, 紫外分光检测RNA纯度与浓度。

1.2.6 水稻原生质体分离

水稻植株在28 ℃黑暗条件下培养2周, 植株长至12~15 cm时, 取叶与茎参照Zhang等[20]的方法提取分离原生质体。

1.2.7 水稻原生质体中H2O2的检测

参照Zhai等[21]的试验方法, 以PEG介导法转化dsRNA或构建的过表达载体进原生质体, 经10 μmol·L-1ABA处理5 min后, 用H2O2荧光探针2′, 7′-二氯荧光素二乙酸酯(2′, 7′-dichlorofluorescein diacetate(H2DCF-DA))标记染色, 在激光共聚焦显微镜下观察。试验数据重复3次, 每个处理至少取60个细胞, 用Zeiss图像软件统计Intensity Mean Value值。利用SPSS statistics 19软件进行统计分析, 以P < 0.05为显著差异。

2 结果与分析 2.1 酵母双杂交筛选SAPK8/9/10与OsRbohB和OsRbohE之间的互作关系

酵母双杂交结果如图 1-AB所示:SD-Trp/-Leu/-His酵母三缺琼脂糖培养基上, 阳性对照生长良好并且变成蓝色, 而阴性对照生长缓慢呈现酵母细胞原本的白色状态。SAPK8/9/10-OsRbohB和SAPK8/9/10-OsRbohE长势良好且呈现蓝色。同时自激活验证结果如图 1-C所示:阳性对照变蓝, pGBKT7-SAPK8/9/10和pGBKT7空载都未变蓝, 说明SAPK8/9/10没有自激活作用。这一结果表明SAPK8/9/10与OsRbohB/E在酵母中存在相互作用。

图 1 SAPK8/9/10与OsRbohB/E酵母双杂交互作筛选 Figure 1 SAPK8/9/10 interact with OsRbohB/E in yeast two-hybrid system A. SAPK8/9/10与OsRbohB酵母双杂交互作筛选; B. SAPK8/9/10与OsRbohE酵母双杂交互作筛选; C. SAPK8/9/10自激活检测。AD:pGAT7 AD载体; BD:pGBKT7载体。 A. SAPK8/9/10 interact with OsRbohB in yeast two-hybrid system; B. SAPK8/9/10 interact with OsRbohE in yeast two-hybrid system; C. Testing SAPK8/9/10 for autoactivation. AD:pGAT7 AD vector; BD:pGBKT7 vector.
2.2 GST-pull down试验体外检测SAPK8/9/10与OsRbohB/E的互作关系

为了证实SAPK8/9/10与OsRbohB/E之间相互作用的真实性, 我们首先利用GST-pull down试验来研究它们之间是否在体外存在直接的物理互作。结果如图 2-AB所示:GST-SAPK8/9/10的泳道经His一抗杂交都出现了目的条带, 而GST空载泳道没有目标条带, 这一结果说明结合GST-SAPK8/9/10的磁珠能够正确捕捉到His-OsRbohB/E, 而GST空载磁珠则不能与His-OsRbohB/E结合。这一结果指出SAPK8/9/10和OsRbohB/E之间在体外存在直接的相互作用。

图 2 GST-pull down验证SAPK8/9/10与OsRbohB/E体外互作 Figure 2 GST-pull down assay for in vitro interaction of SAPK8/9/10 and OsRbohB/E A. GST-pull down验证SAPK8/9/10与OsRbohB(N端)互作; B. GST-pull down验证SAPK8/9/10与OsRbohE(N端)互作。 A. GST-pull down assay for in vitro interaction of SAPK8/9/10 and OsRbohB(N-terminal); B. GST-pull down assay for in vitro interaction of SAPK8/9/10 and OsRbohE(N-terminal).
2.3 SAPK8/9/10与OsRbohB/E在植物体内的相互作用

上述研究表明, SAPK8/9/10与OsRbohB/E在酵母和体外均存在相互作用。然而, 它们之间是否在植物体内也存在相互作用?对此, 本研究以洋葱表皮细胞为材料, 利用BiFC技术验证SAPK8/9/10与OsRbohB/E在植物细胞内是否存在相互作用。如图 3-AB所示:SAPK8/9/10-YFPN与OsRbohB-YFPC组合以及SAPK8/9/10-YFPC与OsRbohE-YFPN组合在激光共聚焦显微镜下可以观察到黄色荧光, 而对照组SAPK8/9/10-YFPN与YFPC组合以及SAPK8/9/10-YFPC与YFPN组合在激光共聚焦显微镜下并没有观察到黄色荧光。这表明SAPK8/9/10与OsRbohB/E之间在植物细胞中存在相互作用。

图 3 在洋葱表皮细胞中BiFC验证SAPK8/9/10与OsRbohB及OsRbohE的相互作用 Figure 3 SAPK8/9/10 interact with OsRbohB/E in vivo as determined by BiFC assay in onion epidermal cells A. BiFC验证SAPK8/9/10与OsRbohB互作; B. BiFC验证SAPK8/9/10与OsRbohE互作。 A. The interaction between SAPK8/9/10 and OsRbohB by BiFC; B. The interaction between SAPK8/9/10 and OsRbohE by BiFC.
2.4 体外磷酸化检测OsRbohB/E与SAPK8/9/10的关系

植物Rboh基因N端含有一段300 bp左右的特异的氨基酸序列, 在Rboh基因调控中起着关键作用CanKaoWenXian_23。因此, 将GST-SAPK8/9/10、His-OsRbohB N端(355个氨基酸)和His-OsRbohE N端(290个氨基酸)原核表达后纯化蛋白, 以Rboh为底物、MBP为阳性对照进行体外免疫沉淀凝胶激酶反应, Typhoon显影结果显示(图 4-AB), SAPK8/9/10能够直接体外磷酸化OsRbohB/E。这一结果表明, OsRbohB/E是SAPK8/9/10的磷酸化底物。

图 4 SAPK8/9/10体外磷酸化OsRbohB/E Figure 4 Phosphorylation of OsRbohB/E by SAPK8/9/10 in vitro A. SAPK8/9/10体外磷酸化OsRbohB; B. SAPK8/9/10体外磷酸化OsRbohE。 A. OsRbohB is phosphorylated by SAPK8/9/10 in vitro; B. OsRbohE is phosphorylated by SAPK8/9/10 in vitro.
2.5 SAPK9/10与OsRbohB/E对ABA诱导的H2O2产生的影响

为了研究ABA诱导下SAPK9/10OsRbohB/E对水稻原生质体中H2O2积累的影响, 利用原生质体瞬时体系, 经10 μmol·L-1ABA处理5 min后用H2O2荧光探针H2DCF-DA标记染色, 在激光共聚焦显微镜下观察。试验结果(图 56)表明:在没有ABA处理下, 与对照组相比, 过表达SAPK9/10OsRbohB/E分别显著增加H2O2荧光强度。在ABA处理后, 与对照组相比, 过表达SAPK9/10OsRbohB/E中荧光强度显著增加。相反, 在没有ABA处理条件下RNAi介导的SAPK9/10OsRbohB/E沉默降低了H2O2荧光强度, ABA处理后荧光强度有所增加, 但是仍然低于对照组荧光强度。由于在试验中未能成功构建SAPK8过表达载体, 因此在后续的试验中没有详细分析SAPK8调节ABA诱导H2O2产生的机制。此外, 过表达SAPKs和沉默OsRbohs原生质体中H2O2荧光强度比过表达OsRbohs和沉默SAPKs原生质体中H2O2荧光强度下降明显, 这一结果说明SAPK9/10通过OsRbohB/E共同调节ABA诱导H2O2产生。

图 5 SAPK10与OsRbohB/E协同调节ABA诱导的H2O2产生 Figure 5 SAPK10 and OsRbohB/E coordinately regulate ABA-induced H2O2 production A、C.SAPK10与OsRbohB共同调节ABA诱导的H2O2产生; B、D.SAPK10与OsRbohE共同调节ABA诱导的H2O2产生。OE10:过表达SAPK 10;OEB:过表达OsRbohB; OEE:过表达OsRbohE; ds10:沉默SAPK 10;dsB:沉默OsRbohB; dsE:沉默OsRbohE。标尺=50 μm。 A, C.SAPK10 and OsRbohB co-regulated ABA-induced H2O2 production; B, D.SAPK10 and OsRbohE co-regulated ABA-induced H2O2 production. OE10:Over-expression of SAPK 10;OEB:Over-expression of OsRbohB; OEE:Over-expression of OsRbohE; ds10:Silencing of SAPK 10;dsB:Silencing of OsRbohB; dsE:Silencing of OsRbohE. Bar=50 μm.
图 6 SAPK9与OsRbohB/E协同调节ABA诱导的H2O2产生 Figure 6 SAPK9 and OsRbohB/E coordinately regulate ABA-induced H2O2 production A、C.SAPK9与OsRbohB共同调节ABA诱导的H2O2产生; B、D.SAPK9与OsRbohE共同调节ABA诱导的H2O2产生。OE9:过表达SAPK 9;OEB:过表达OsRbohB; OEE:过表达OsRbohE; ds9:沉默SAPK 9;dsB:沉默OsRbohB; dsE:沉默OsRbohE。标尺=50 μm。 A, C.SAPK9 and OsRbohB co-regulated ABA-induced H2O2 production; B, D.SAPK9 and OsRbohE co-regulated ABA-induced H2O2 production. OE9:Over-expression of SAPK 9;OEB:Over-expression of OsRbohB; OEE:Over-expression of OsRbohE; ds9:Silencing of SAPK 9;dsB:Silencing of OsRbohB; dsE:Silencing of OsRbohE. Bar=50 μm.
3 讨论

ABA信号转导可以分成3个阶段:ABA的产生与运输; ABA信号的感知与传递; ABA信号的应答与调控。其中ABA的信号感知与传递系统包含的核心信号成员主要有PYR/RCAR等ABA的受体家族、PP2C家族以及SnRK2激酶家族等[15, 24-25]。PP2C作为一种负调控元件, 在正常条件下, PP2C与SnRK2结合, 去磷酸化SnRK2, 从而导致SnRK2失活, 不能将信号向下游传递。当植物受到外界胁迫时, ABA迅速合成并积累, 结合了ABA的PYR/PYL/RCAR与PP2Cs相互作用能够抑制PP2C的活性[26], 从而解除PP2C对SnRK2活性的抑制。处于激活状态的SnRK2可以将ABA信号传递给下游组分, 包括通过蛋白磷酸化作用激活下游转录因子(如AREBl/ABF2、AREB2/ABF4和ABF3), 正向调控ABA信号应答基因的表达[27-28], 以及直接作用于相关膜蛋白(如阴离子通道和钾离子通道), 最终诱导气孔关闭[29-30]

研究发现, 拟南芥OST1能够直接与NADPH氧化酶中的AtrbohF互作, 且AtrbohF被OST1磷酸化, AtrbohF中的Ser13和Ser174残基是其磷酸化位点[16]。水稻中与拟南芥OST1同源的SAPK8、SAPK9和SAPK10激酶活性均可被ABA上调[17], 且OsRbohBOsRbohE被显示受ABA诱导表达上调, 暗示这些基因参与ABA诱导的H2O2产生。那么, 水稻中这些SAPKs是否与OsRbohs之间均存在互作关系?为此, 本研究先利用酵母双杂交系统进行了初步的互作探究, 结果显示SAPK8、SAPK9和SAPK10均与OsRbohB以及OsRbohE存在相互作用关系。然后又利用GST-pull down技术以及双分子荧光互补(BiFC)技术证实了SAPK8/9/10与OsRbohB/E在体内、外存在相互作用。同时, 体外磷酸化技术分析显示, SAPK8/9/10能够磷酸化OsRbohB/E, 即OsRbohB/E均可作为SAPK8/9/10磷酸化底物。这些结果暗示, 在ABA信号通路中, SAPK8/9/10与OsRbohB/E可能通过互作发挥作用。

在拟南芥中SnRK2家族蛋白激酶OST1(SnRK2.6)作用于NADPH氧化酶AtrbohF, 催化胞外活性氧产生, 放大ROS信号, 引起ROS介导的信号级联反应[16]。那么, 水稻中这些SAPKs与OsRbohs在ABA信号转导中是否共同参与调节ABA诱导的H2O2的产生?本试验中, 在SAPKs-OE、OsRbohs-OE原生质体中H2O2的荧光强度均上调, 且ABA处理后上调趋势进一步增强; 在ds-SAPKs、ds-OsRbohs原生质体中H2O2的荧光强度显著下降, ABA诱导的H2O2反而被抑制。与单独过表达SAPKsOsRbohs相比, 过表达SAPKs且沉默OsRbohs的原生质体以及过表达OsRbohs且沉默SAPKs的原生质体中H2O2产生均显著降低, ABA诱导的H2O2反而被阻止。这些结果表明:在ABA信号转导中, 处于激活状态的SAPK9/10与OsRbohB/E相互作用, 使NADPH氧化酶活性升高, 进而诱导H2O2的积累。

综上所述, 本试验证实SAPK8/9/10与OsRbohB以及OsRbohE在体内、外存在相互作用, OsRbohs是SAPKs磷酸化的底物, SAPKs与OsRbohs协同调节ABA诱导的H2O2产生。因此, 我们推测在水稻ABA响应干旱胁迫产生H2O2的过程中可能存在这样一条通路, 即干旱诱导的ABA积累激活SAPKs, 活化的SAPKs磷酸化OsRbohs, 进而诱导H2O2的产生。本文为进一步研究ABA信号通路在植物响应干旱胁迫的作用机制中提供了理论依据。然而, OsRbohs作为SAPKs磷酸化的底物, 其磷酸化位点还需进一步的研究来证实。此外, ABA信号通路中SAPKs与OsRbohs是通过单独互作还是协同互作在ABA诱导H2O2产生过程中发挥作用, 这也是之后研究中需要进一步解决的问题。

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