A cotton mitogen-activated protein kinase （GhMPK6） is involved in ABA-induced CAT1 expression and H2O2 production

The mitogen-activated protein kinase (MAPK) cascade is one of the major and evolutionally conserved signaling pathways and plays a pivotal role in the regulation of stress and developmental signals in plants.Here,we identified one gene,GhMPK6,encoding an MAPK protein in cotton.GFP fluorescence assay demonstrated that GhMAPK6 is a cytoplasm localized protein.Quantitative RT-PCR analysis revealed that mRNA accumulation of GhMPK6 was significantly promoted by abscisic acid (ABA).Overexpression of GhMPK6 gene in the T-DNA insertion mutant atmkkl (SALK_015914) conferred a wild-type phenotype to the transgenic plants in response to ABA.Under ABA treatment,cotyledon greening/expansion in GhMPK6 transgenic lines and wild type was significantly inhibited,whereas the atmkkl mutant showed a relatively high cotyledon greening/expansion ratio.Furthermore,CAT1 expression and H2O2 levels in leaves of GhMPK6 transgenic lines and wild type were remarkably higher than those of atmkkl mutant with ABA treatment.Collectively,our results suggested that GhMPK6 may play an important role in ABA-induced CAT1 expression and H2O2 production.     

The AtMKK3 pathway mediates ABA and salt signaling in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Mitogen-activated protein (MAP) kinases cascades mediate cellular responses to a great variety of different extracellular signals in plants. Activation of a MAP kinase occurs after phosphorylation by an upstream dual-specificity protein kinase, known as a MAP kinase kinase. However, only a few of the MAPK kinases in Arabidopsis have been investigated. An active AtMKK3, 35S:AtMPK1, 35S:AtMPK2, and 35S:AtMPK3 constructs were built and their transformed plants were generated. The kinase activity of AtMPK1 or AtMPK2 was stimulated by active AtMKK3 in transient analysis of tobacco leaves. Coimmunoprecipitation experiments indicated interaction between AtMKK3 and AtMPK1 or AtMPK2 in the coexpressed tissues of AtMKK3 and AtMPK1 or AtMKK3 and AtMPK2. RT-PCR analysis showed that AtMKK3 and AtMPK1, or AtMKK3 and AtMPK2 were co-expressed in diverse plant tissues. Plants overexpressing AtMKK3 exhibited an enhanced tolerance to salt and were more sensitive to ABA. Plants overexpressing AtMPK1 or AtMPK2 were also more sensitive to ABA. AtMPK1 or AtMPK2 can be activated by cold, salt, and ABA. AtMKK3, AtMPK1, and AtMPK2 genes were induced by ABA or stress treatments. All these data indicated that the ABA signal transmitted to a MAPK kinase signaling cascade and could be amplified through MAP kinase1 or MAP kinase2 for increasing salt stress tolerance in Arabidopsis.     

AtMKK1 and AtMPK6 are involved in abscisic acid and sugar signaling in <Emphasis Type="Italic">Arabidopsis</Emphasis> seed germination

Abscisic acid (ABA) and sugars have been well established to be crucial factors controlling seed germination of Arabidopsis. Here we demonstrate that AtMKK1 and AtMPK6 are both critical signals involved in ABA and sugar-regulated seed germination. Wild type plants depended on stratification and after-ripening for seed germination, whereas this dependence on either stratification or after-ripening was not required for mutants of mkk1 and mpk6 as well as their double mutant mkk1 mpk6. While seed germination of wild type plants was sensitively inhibited by ABA and glucose, mkk1, mpk6 and mkk1 mpk6 were all strongly resistant to ABA or glucose treatments, and in contrast, plants overexpressing MKK1 or MPK6 were super-sensitive to ABA and glucose. Glucose treatment significantly induced increases in MKK1 and MPK6 activities. These results clearly indicate that MKK1 and MPK6 are involved in the ABA and sugar signaling in the process of seed germination. Further experiments showed that glucose was capable of inducing ABA biosynthesis by up-regulating NCED3 and ABA2, and furthermore, this up-regulation of NCED3 and ABA2 was arrested in the mkk1 mpk6 double mutant, indicating that the inhibition of seed germination by glucose is potentially resulted from sugar-induced up-regulation of the ABA level. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Harpin induces activation of the Arabidopsis mitogen-activated protein kinases AtMPK4 and AtMPK6

Mitogen-activated protein kinases (MAPKs) are key enzymes that mediate adaptive responses to various abiotic and biotic stresses, including pathogen challenge. The proteinaceous bacterial elicitor harpin (secreted by Pseudomonas syringae pv syringae) activates two MAPKs in suspension cultures of Arabidopsis var. Landsberg erecta. In this study, we show that harpin and exogenous hydrogen peroxide (H(2)O(2)) activate myelin basic protein kinases in Arabidopsis leaves. Using anti-AtMPK4 and anti-AtMPK6 antibodies, we identify the harpin-activated MAPKs in both leaves and suspension cultures as AtMPK4 and AtMPK6, and show that H(2)O(2), generated by Arabidopsis cells in response to challenge with harpin, activates only AtMPK6. However, treatments with catalase, which removes H(2)O(2), or diphenylene iodonium, which inhibits superoxide and H(2)O(2) production, do not inhibit harpin-induced activation of AtMPK4 or AtMPK6. In addition, activation of AtMPK4 but not AtMPK6 is inhibited by the MAPK kinase inhibitor PD98059. Neither harpin nor H(2)O(2) has any effect on AtMPK4 or AtMPK6 gene expression. In addition, the expression of AtMEKK1, AtMEK1, or AtMKK2, previously shown to be potential functional partners of AtMPK4, were not affected by either harpin or H(2)O(2) treatments. These data suggest that harpin activates several signaling pathways, one leading to stimulation of the oxidative burst and others leading to the activation of AtMPK4 or AtMPK6.     

AtMEK1 mediates stress-induced gene expression of CAT1 catalase by triggering H2O2 production in Arabidopsis

Catalase and hydrogen peroxide (H(2)O(2)) have been extensively studied for their roles in various stress responses. However, little is known about the triggering mechanisms for stress-induced catalase gene expression or about H(2)O(2) production as a stress signal. It is reported here that ABA-, drought-, and salt stress-induced gene expression of CAT1 catalase is mediated by AtMEK1, an Arabidopsis MAPK kinase, by triggering H(2)O(2) signal production. Both CAT1 expression and AtMEK1 activity were activated by ABA, drought, and salt stresses. The mek1 mutant totally blocked stress-induced CAT1 expression and, interestingly, stress-induced H(2)O(2) production was also blocked. Over-expression of AtMEK1 significantly promoted stress-induced CAT1 expression, and also promoted H(2)O(2) production. These results conclusively indicate that stress-induced CAT1 expression is mediated by AtMEK1 and, furthermore, that the triggering of H(2)O(2) production might be involved in this process, as further proved by the observation that CAT1 expression was induced by applied H(2)O(2.) Surprisingly, the signalling mechanisms for stress-induced gene expression of CAT2 and CAT3 were very different from that of CAT1. Except for drought stress, expression of CAT2 or CAT3 was also activated by salt stress or ABA treatment, and AtMEK1 was not proved to be involved in the drought-induced expression of CAT2 or CAT3. Further studies showed that stomatal movement was much less sensitive to ABA in AtMEK1 mutant (mek1), and over-expression of AtMEK1 in Arabidopsis increased plant resistance to drought or salt stress, which further demonstrated that AtMEK1 is a crucial mediator in plant stress signal transduction.     

AtMPK4 is required for male-specific meiotic cytokinesis in Arabidopsis

Mitogen-activated protein kinase (MAPK) cascades have been implicated in regulating various aspects of plant development, including somatic cytokinesis. The evolution of expanded plant MAPK gene families has enabled the diversification of potential MAPK cascades, but functionally overlapping components are also well documented. Here we report that Arabidopsis MPK4, an MAPK that was previously described as a regulator of disease resistance, can interact with and be phosphorylated by the cytokinesis-related MAP kinase kinase, AtMKK6. In mpk4 mutant plants, anthers can develop normal microspore mother cells (MMCs) and peripheral supporting tissues, but the MMCs fail to form a normal intersporal callose wall after male meiosis, and thus cannot complete meiotic cytokinesis. Nevertheless, the multinucleate mpk4 microspores subsequently proceed through mitotic cytokinesis, resulting in enlarged mature pollen grains that possess increased sets of the tricellular structure. This pollen development phenotype is reminiscent of those observed in both atnack2/tes/stud and anq1/mkk6 mutants, and protein-protein interaction analysis defines a putative signalling module linking AtNACK2/TES/STUD, AtANP3, AtMKK6 and AtMPK4 together as a cascade that facilitates male-specific meiotic cytokinesis in Arabidopsis.     

Activation of the NaCl- and drought-induced RD29A and RD29B promoters by constitutively active Arabidopsis MAPKK or MAPK proteins

Mitogen-activated protein (MAP) kinases mediate cellular responses to a wide variety of stimuli. Activation of a MAP kinase (MAPK) occurs after phosphorylation by an upstream MAP kinase kinase (MAPKK). The Arabidopsis thaliana genome encodes 10 MKKs, but few of these have been shown directly to activate any of the 20 Arabidopsis MAPKs (AtMPKs) and NaCl-, drought- or abscisic acid (ABA)-induced genes RD29A or RD29B. We have constructed the constitutively activated form for nine of the 10 AtMKK proteins, and tested their ability to activate the RD29A and RD29B promoters and also checked the ability of the nine activated AtMKK proteins to phosphorylate 11 of the AtMPK proteins in transient assays. The results show that three proteins, AtMKK1, AtMKK2 and AtMKK3, could activate the RD29A promoter, while these three and two additional AtMKK6/8 proteins could activate the RD29B promoter. Four other proteins, AtMKK7/AtMKK9 and AtMKK4/AtMKK5, can cause hypersensitive response (HR) in tobacco leaves using transient analysis. The activation of the RD29A promoter correlated with four uniquely activated AtMPK proteins. A novel method of activating AtMPK proteins by fusion to a cis-acting mutant of a human MAPK kinase MEK1 was used to confirm that specific members of the AtMPK gene family can activate the RD29A stress pathway.     

Knockout of AtMKK1 enhances salt tolerance and modifies metabolic activities in Arabidopsis

Mitogen-activated protein kinase (MAPK) pathways represent a crucial regulatory mechanism in plant development. The ability to activate and inactivate MAPK pathways rapidly in response to changing conditions helps plants to adapt to a changing environment. AtMKK1 is a stress response kinase that is capable of activating the MAPK proteins AtMPK3, AtMPK4 and AtMPK6. To elucidate its mode of action further, several tests were undertaken to examine the response of AtMKK1 to salt stress using a knockout (KO) mutant of AtMKK1. We found that AtMKK1 mutant plants tolerated elevated levels of salt during both germination and adulthood. Proteomic analysis indicated that the level of the α subunit of mitochrondrial H⁺-ATPase, mitochrondial NADH dehydrogenase and mitochrondrial formate dehydrogenase was enhanced in AtMKK1 knockout mutants upon high salinity stress. The level of formate dehydrogenase was further confirmed by immunoblotting and enzyme assay. The possible involvement of these enzymes in salt tolerance is discussed.     

MEKK1, MKK1/MKK2 and MPK4 function together in a mitogen-activated protein kinase cascade to regulate innate immunity in plants

Mitogen-activated protein kinase (MAPK) cascades play important roles in regulating plant innate immune responses. In a genetic screen to search for mutants with constitutive defense responses, we identified multiple alleles of mpk4 and mekk1 that exhibit cell death and constitutive defense responses. Bimolecular fluorescence complementation (BiFC) analysis showed that both MPK4 and MEKK1 interact with MKK1 and MKK2, two closely related MAPK kinases. mkk1 and mkk2 single mutant plants do not have obvious mutant phenotypes. To test whether MKK1 and MKK2 function redundantly, mkk1 mkk2 double mutants were generated. The mkk1 mkk2 double mutant plants die at seedling stage and the seedling-lethality phenotype is temperature-dependent. Similar to the mpk4 and mekk1 mutants, the mkk1 mkk2 double mutant seedlings accumulate high levels of H2O2, display spontaneous cell death, constitutively express Pathogenesis Related (PR) genes and exhibit pathogen resistance. In addition, activation of MPK4 by flg22 is impaired in the mkk1 mkk2 double mutants, suggesting that MKK1 and MKK2 function together with MPK4 and MEKK1 in a MAP kinase cascade to negatively regulate innate immune responses in plants.     

Mitogen-activated protein kinase is involved in abscisic acid-induced antioxidant defense and acts downstream of reactive oxygen species production in leaves of maize plants

The role of mitogen-activated protein kinase (MAPK) in abscisic acid (ABA)-induced antioxidant defense was investigated in leaves of maize (Zea mays) plants. Treatments with ABA or H(2)O(2) induced the activation of a 46-kD MAPK and enhanced the expression of the antioxidant genes CAT1, cAPX, and GR1 and the total activities of the antioxidant enzymes catalase, ascorbate peroxidase, glutathione reductase, and superoxide dismutase. Such enhancements were blocked by pretreatment with several MAPK kinase inhibitors and reactive oxygen species inhibitors or scavengers. Pretreatment with MAPK kinase inhibitors also substantially arrested the ABA-induced H(2)O(2) production after 2 h of ABA treatment, but did not affect the levels of H(2)O(2) within 1 h of ABA treatment. Pretreatment with several inhibitors of protein tyrosine phosphatase, which is believed to be a negative regulator of MAPK, only slightly prevented the ABA-induced H(2)O(2) production, but did not affect the ABA-induced MAPK activation and ABA-enhanced antioxidant defense systems. These results clearly suggest that MAPK but not protein tyrosine phosphatase is involved in the ABA-induced antioxidant defense, and a cross talk between H(2)O(2) production and MAPK activation plays a pivotal role in the ABA signaling. ABA-induced H(2)O(2) production activates MAPK, which in turn induces the expression and the activities of antioxidant enzymes. The activation of MAPK also enhances the H(2)O(2) production, forming a positive feedback loop.     

AtMKK6 and AtMPK13 are required for lateral root formation in Arabidopsis

The mitogen-activated protein (MAP) kinase cascades are important signaling components that mediate various biological pathways in all eukaryotic cells. In our recent publication,¹ we identified AtMPK4 as one of the downstream targets of AtMKK6 that is required for executing male-specific meiotic cytokinesis. Here we provide evidence that another target, AtMPK13, is developmentally co-expressed with AtMKK6 in Arabidopsis, and both AtMPK13 and AtMKK6 display high Promoter::GUS activity in the primary root tips and at the lateral root primordia. Partial suppression of either AtMKK6 or AtMPK13 expression significantly reduces the number of lateral roots in the transgenic lines, suggesting that the AtMKK6-AtMPK13 module positively regulates lateral root formation.Key words: MAP kinase modules, lateral root, RNAi, developmental specificity, pericycle     

共聚焦显微技术研究ABA诱导蚕豆气孔保卫细胞H2O2的产生（简报）

The methods of confocal laser scanning microscopy (CLSM) and microinjection were used to study ABA-induced H2O2 in guard cells (Vicia faba), which were labeled with H2O2 specific probe-2, 7-dichlorofluorescin diacetate(H2DCFDA). The results indicated 100 U/mL catalase (CAT) could inhibit partly stomatal closure induced by ABA. 10(-3) mmol/L ABA could significantly induce H2O2 production in chloroplast in guard cells of Vicia faba following microinjection, and 100 U/mL CAT could partly abolish the effects following simultaneous microinjection of ABA and CAT. These suggest that H2O2 is possibly involved in ABA signaling leading to stomatal closure.     

共聚焦显微技术研究ABA诱导蚕豆气孔保卫细胞H_2O_2的产生(简报)

逆境下,植物细胞内ABA含量急剧增加,同时植物也可通过一些酶代谢反应积累活性氧,如H_2O_2,O_2~-。ABA作为逆境信号对气孔运动的显著调节作用已被诸多实验所证实,但关于其对气孔运动调节的细节还知之甚少。H_2O_2作为氧化信号分子在植物抗病信号转导中已得到广泛研究,但H_2O_2是否介导保卫细胞的气孔运动还缺乏直接的证据。我们已初步发现H_2O_2可参与外源ABA诱     

Diverse stress signals activate the C1 subgroup MAP kinases of Arabidopsis

Mitogen-activated protein kinase (MAPK) cascades play an important role in mediating stress responses in plants. In Arabidopsis, 20 MAPKs have been identified and classified into four major groups (A-D). Little is known about the role of group C MAPKs. We have studied the activation of Arabidopsis subgroup C1 MAPKs (AtMPK1/AtMPK2) in response to mechanical injury. An increase in their kinase activity was detected in response to wounding that was blocked by cycloheximide. Jasmonic acid (JA) activated AtMPK1/AtMPK2 in the absence of wounding. Wound and JA-induction of AtMPK1/2 kinase activity was not prevented in the JA-insensitive coi1 mutant. Other stress signals, such as abscisic acid (ABA) and hydrogen peroxide, activated AtMPK1/2. This report shows for the first time that regulation of AtMPK1/2 kinase activity in Arabidopsis might be under the control of signals involved in different kinds of stress.     

Abscisic Acid Activation of Plasma Membrane Ca2+ Channels in Guard Cells Requires Cytosolic NAD(P)H and Is Differentially Disrupted Upstream and Downstream of Reactive Oxygen Species Production in abi1-1 and abi2-1 Protein Phosphatase 2C Mutants

The hormone abscisic acid (ABA) regulates stress responses and developmental processes in plants. Calcium-permeable channels activated by reactive oxygen species (ROS) have been shown recently to function in the ABA signaling network in Arabidopsis guard cells. Here, we report that ABA activation of these I(Ca) Ca(2)+ channels requires the presence of NAD(P)H in the cytosol. The protein phosphatase 2C (PP2C) mutant abi1-1 disrupted ABA activation of I(Ca) channels. Moreover, in abi1-1, ABA did not induce ROS production. Consistent with these findings, in abi1-1, H(2)O(2) activation of I(Ca) channels and H(2)O(2)-induced stomatal closing were not disrupted, suggesting that abi1-1 impairs ABA signaling between ABA reception and ROS production. The abi2-1 mutation, which lies in a distinct PP2C gene, also disrupted ABA activation of I(Ca). However, in contrast to abi1-1, abi2-1 impaired both H(2)O(2) activation of I(Ca) and H(2)O(2)-induced stomatal closing. Furthermore, ABA elicited ROS production in abi2-1. These data suggest a model with the following sequence of events in early ABA signal transduction: ABA, abi1-1, NAD(P)H-dependent ROS production, abi2-1, I(Ca) Ca(2)+ channel activation followed by stomatal closing.     

The Arabidopsis thaliana MEK AtMKK6 activates the MAP kinase AtMPK13

Mitogen-activated protein (MAP) kinases mediate cellular responses to a wide variety of stimuli. Activation of a MAP kinase occurs after phosphorylation by an upstream dual-specificity protein kinase, known as a MAP kinase kinase or MEK. The Arabidopsis thaliana genome encodes 10 MEKs but few of these have been shown directly to activate any of the 20 Arabidopsis MAP kinases. We show here that functional complementation of the cell lysis phenotype of a mutant yeast strain depends on the co-expression of the Arabidopsis MEK AtMKK6 and the MAP kinase AtMPK13. The kinase activity of AtMPK13 is stimulated in the presence of AtMKK6 in yeast cells. RT-PCR analysis showed the co-expression of these two genes in diverse plant tissues. These data show that AtMKK6 can functionally activate the MAP kinase AtMPK13.