蛋白激酶组在玉米叶片ABA和H2O2诱导抗氧化防护中的作用

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66kDa, 52kDa, 49kDa和35kDa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66kDa和49kDa的蛋白激酶可能在ROS的下游起作用, 而52kDa和35kDa的蛋白激酶可能在ABA和ROS的下游起作用。     

蛋白激酶组在玉米叶片ABA和H202诱导抗氧化防护中的作用

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ca^2＋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66kDa,52kDa,49kDa和35kDa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66kDa和49kDa的蛋白激酶可能在ROS的下游起作用,而52kDa和35kDa的蛋白激酶可能在ABA和ROS的下游起作用。     

一氧化氮在脱落酸诱导的玉米叶片亚细胞抗氧化防护中的作用

研究了ABA诱导NO产生的来源以及NO在ABA诱导的玉米叶片H2O2累积和亚细胞水平抗氧化中的作用。ABA诱导玉米叶片NO的产生以及NOS活性增加,NOS抑制剂抑制这种增加。NO清除剂和NR抑制剂预处理也抑制了ABA诱导的NO产生,但是并不影响ABA诱导的NOS活性,结果提示了ABA诱导的NO的产生来源于NOS和NR2条途径。NO清除剂、NOS抑制剂和NR抑制剂预处理抑制了ABA和H2O2诱导的抗氧化防护酶基因SOD4、cAPX、GR1的表达和叶绿体及细胞溶质抗氧化酶活性的增加,表明NO参与ABA和H2O2诱导的玉米亚细胞抗氧化防护系统。另一方面,以NO供体SNP预处理减少了ABA诱导的H2O2的累积,而c—PTIO逆转了SNP减少ABA诱导的H2O2累积的作用。SNP处理诱导了亚细胞抗氧化酶活性的增加,用c—PTIO预处理抑制了这种增加。实验结果表明ABA诱导H2O2和INO产生,NO上调了玉米亚细胞抗氧化防护酶活性,进而防止玉米叶片中H2O2的过量累积。因此在玉米ABA诱导的信号转导中有一个NO和H2O2负反馈环。     

SB202190 调节蚕豆保卫细胞中SA 诱导H2O2 产生

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(mitogenactivated 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p38MAP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。     

SB202190调节蚕豆保卫细胞中SA诱导H2O2产生

运用激光共聚焦扫描技术,在p38MAP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：p38MAP激酶专一抑制剂SB202190处理没有导致蚕豆保卫细胞中H2O2和Ca^2＋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Ca^2＋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Ca^2＋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p38MAP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。     

盐胁迫对玉米根尖质膜上受钙激活的蛋白激酶的影响

以02mal／L的NaCl对玉米根尖进行盐胁迫,导致质膜上一种受钙激活的蛋白激酶（该激酶表现为钙依赖蛋白激酶的特性）的活性迅速增加。盐胁迫至15min时激酶的活性达到最大,较对照高30％,以后逐渐降低,盐胁迫至切50min时激酶活性仍略高于对照。10％PEG6000处理玉米很尖也可诱导质膜上受钙激活的蛋白激酶活性增加。用外源ABA处理玉米根尖,未导致上述蛋白激酶活性的变化。放射自显影显示质膜上33kD和58kD两种蛋白可能是质膜受钙激活的蛋白激酶的内源底物。     

H_2O_2和MAPK介导油菜素内酯诱导番茄抗氧化防护酶SOD和CAT的信号途径

本文以番茄为材料,研究H2O2和MAPK在BR诱导的抗氧化防护系统中的作用。结果表明,外源BR提高抗氧化防护酶SOD和CAT活性;而这种诱导机制被H2O2产生抑制剂二苯基碘（DPI）和MEK1/2专一抑制剂PD98059阻断。进一步研究发现：BR能够诱导细胞质外体H2O2的产生,这种诱导被PD98059抑制;BR能够活化一种49kDaMAPK,这种活化被DPI抑制。本研究结果证实细胞质外体H2O2和MAPK候选激酶（49kDaMAPK）信号参与BR诱导的抗氧化防酶途径,且两者之间存在交互作用。     

五味子对H_2O_2诱导的HaCaT细胞氧化应激损伤的保护作用

检测五味子不同组分对H2O2诱导的HaCaT细胞氧化应激损伤的保护作用,筛选得到其活性组分。利用大孔吸附树脂法,对五味子果实提取物进行分离纯化,得到不同的组分段。以DPPH自由基清除能力和总抗氧化能力(ABTS法)为考察指标,对五味子各组分的抗氧化活性进行评价;采用MTT法筛选五味子各组分对H2O2诱导的HaCaT细胞氧化应激损伤的保护作用,获得活性组分。结果显示,50%乙醇洗脱物抗氧化活性最强,能提高H2O2处理后的HaCaT细胞存活率,减少MDA的生成,上调SOD酶和GSH酶活性。研究结果证实,50%乙醇洗脱物是五味子保护皮肤细胞免受H2O2诱导氧化应激的活性组分。     

ABA诱导玉米叶质外体H2O2积累的机制

通过组织化学染色和电镜观察并结合酶活性分析表明,ABA可通过诱导玉米（Zea mays L、）叶片质膜NADPH氧化酶、细胞壁POD及质外体PAO活性的升高,使其质外体产生H2O2;其中质膜NADPH氧化酶起主要作用。     

苹果和葡萄果实蛋白激酶特性分析

以组蛋白Ⅲ S作苹果和葡萄果肉蛋白激酶制剂底物时 ,反应体系中加EGTA可抑制蛋白激酶活性 ,而加Ca2 可激活蛋白激酶的活性 ,表明苹果和葡萄果实中有依赖钙的蛋白激酶存在。而且 ,葡萄果实微粒体蛋白激酶呈热稳定性 ,苹果果实微粒体蛋白激酶对热敏感。以髓鞘碱性蛋白 (MBP)作底物 ,在苹果和葡萄果实微粒体中都检测出很高的蛋白激酶活性 ,并且不依赖于钙 ,说明苹果和葡萄果实中可能有分裂原激活的蛋白激酶 (MAP激酶 )的存在。苹果和葡萄果实MAP激酶的活性都表现出对二价阳离子Mg2 或Mn2 的依赖 ,并对高温处理表现出了激活效应     

Role of Protein Kinases in Abscisic Acid and H2O2 Induced Antioxidant Defense in Maize

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 kinases and calcium dependent protein kinases in the leaves of maize seedlings. However, ABA-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 -induced kinase activity but also ABA or H2O2-induced antioxidant enzyme activity. Protein phosphorylation caused by ABA and H2O2 preceded ABA or H2O2 -induced antioxidant defense obviously. Using in-gel kinase assays, our results showed that several protein kinases with molecular masses of 66kDa, 52kDa, 49kDa and 35kDa respectively might mediate ABA and H2O2-induced antioxidant defense. And the 66kDa and 49kDa protein kinases may act downstream of ROS, and the 52kDa and 35kDa protein kinases may act between ABA and ROS in ABA-induced antioxidant defensive signaling.     

Differential abscisic acid regulation of guard cell slow anion channels in Arabidopsis wild-type and abi1 and abi2 mutants.

Abscisic acid (ABA) regulates vital physiological responses, and a number of events in the ABA signaling cascade remain to be identified. To allow quantitative analysis of genetic signaling mutants, patch-clamp experiments were developed and performed with the previously inaccessible Arabidopsis guard cells from the wild type and ABA-insensitive (abi) mutants. Slow anion channels have been proposed to play a rate-limiting role in ABA-induced stomatal closing. We now directly demonstrate that ABA strongly activates slow anion channels in wild-type guard cells. Furthermore, ABA-induced anion channel activation and stomatal closing were suppressed by protein phosphatase inhibitors. In abi1-1 and abi2-1 mutant guard cells, ABA activation of slow anion channels and ABA-induced stomatal closing were abolished. These impairments in ABA signaling were partially rescued by kinase inhibitors in abi1 but not in abi2 guard cells. These data provide cell biological evidence that the abi2 locus disrupts early ABA signaling, that abi1 and abi2 affect ABA signaling at different steps in the cascade, and that protein kinases act as negative regulators of ABA signaling in Arabidopsis. New models for ABA signaling pathways and roles for abi1, abi2, and protein kinases and phosphatases are discussed.     

Protein Phosphatases 2C Regulate the Activation of the Snf1-Related Kinase OST1 by Abscisic Acid in Arabidopsis

The plant hormone abscisic acid (ABA) orchestrates plant adaptive responses to a variety of stresses, including drought. This signaling pathway is regulated by reversible protein phosphorylation, and genetic evidence demonstrated that several related protein phosphatases 2C (PP2Cs) are negative regulators of this pathway in Arabidopsis thaliana. Here, we developed a protein phosphatase profiling strategy to define the substrate preferences of the HAB1 PP2C implicated in ABA signaling and used these data to screen for putative substrates. Interestingly, this analysis designated the activation loop of the ABA activated kinase OST1, related to Snf1 and AMPK kinases, as a putative HAB1 substrate. We experimentally demonstrated that HAB1 dephosphorylates and deactivates OST1 in vitro. Furthermore, HAB1 and the related PP2Cs ABI1 and ABI2 interact with OST1 in vivo, and mutations in the corresponding genes strongly affect OST1 activation by ABA. Our results provide evidence that PP2Cs are directly implicated in the ABA-dependent activation of OST1 and further suggest that the activation mechanism of AMPK/Snf1-related kinases through the inhibition of regulating PP2Cs is conserved from plants to human.     

Differential activation of the rice sucrose nonfermenting1-related protein kinase2 family by hyperosmotic stress and abscisic acid

To date, a large number of sequences of protein kinases that belong to the sucrose nonfermenting1-related protein kinase2 (SnRK2) family are found in databases. However, only limited numbers of the family members have been characterized and implicated in abscisic acid (ABA) and hyperosmotic stress signaling. We identified 10 SnRK2 protein kinases encoded by the rice (Oryza sativa) genome. Each of the 10 members was expressed in cultured cell protoplasts, and its regulation was analyzed. Here, we demonstrate that all family members are activated by hyperosmotic stress and that three of them are also activated by ABA. Surprisingly, there were no members that were activated only by ABA. The activation was found to be regulated via phosphorylation. In addition to the functional distinction with respect to ABA regulation, dependence of activation on the hyperosmotic strength was different among the members. We show that the relatively diverged C-terminal domain is mainly responsible for this functional distinction, although the kinase domain also contributes to these differences. The results indicated that the SnRK2 protein kinase family has evolved specifically for hyperosmotic stress signaling and that individual members have acquired distinct regulatory properties, including ABA responsiveness by modifying the C-terminal domain.     

Catalytic mechanism and kinase interactions of ABA-signaling PP2C phosphatases

Abscisic acid (ABA) is an essential hormone that controls plant growth, development and responses to abiotic stresses. ABA signaling is mediated by type 2C protein phosphatases (PP2Cs), including HAB1 and ABI2, which inhibit stress-activated SnRK2 kinases and whose activity is regulated by ABA and ABA receptors. Based on biochemical data and our previously determined crystal structures of ABI2 and the SnRK2.6–HAB1 complex, we present the catalytic mechanism of PP2C and provide new insight into PP2C–SnRK2 interactions and possible roles of other SnRK2 kinases in ABA signaling.     

ABA-activated SnRK2 protein kinase is required for dehydration stress signaling in Arabidopsis

Protein phosphorylation has pivotal roles in ABA and osmotic stress signaling in higher plants. Two protein phosphatase genes, ABI1 and ABI2, are known to regulate these signaling pathways in Arabidopsis: The identity of ABA-activated protein kinases required for the ABA signaling, however, remains to be elucidated. Here we demonstrate that two protein kinases, p44 and p42, were activated by ABA in Arabidopsis T87 cultured cells, and at least one protein kinase, p44, was activated not only by ABA but also by low humidity in Arabidopsis plants. Analysis of T-DNA knockout mutants and biochemical analysis using a specific antibody revealed that the p44 is encoded by a SnRK2-type protein kinase gene, SRK2E. The srk2e mutation resulted in a wilty phenotype mainly due to loss of stomatal closure in response to a rapid humidity decrease. ABA-inducible gene expression of rd22 and rd29B was suppressed in srk2e. These results show that SRK2E plays an important role in ABA signaling in response to water stress.