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.     

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.     

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     

Quantitative proteomics identifies oxidant-induced,AtMPK6-dependent changes in Arabidopsis thaliana protein profiles

In Arabidopsis thaliana, oxidant-induced signalling has been shown to utilize the mitogen-activated protein kinase (MAPK), AtMPK6. To identify proteins whose accumulation is altered by ozone in an AtMPK6-dependent manner we employed isotope-coded affinity tagging (ICAT) technology to investigate the impact of AtMPK6-suppression on the protein profiles in Arabidopsis both before (air control) and during continuous ozone (O₃) fumigation (500 nL L⁻¹ for 8 h). Among the 150 proteins positively identified and quantified in the O₃-treated plants, we identified thirteen proteins whose abundance was greater in the AtMPK6-suppressed genotype than in wild-type (WT). These include the antioxidant proteins, monodehydroascorbate reductase, peroxiredoxin Q, and glutathione reductase. A further eighteen proteins were identified whose abundance was lower in the ozone-treated AtMPK6-suppressed line relative to ozone-exposed WT plants. These predominantly comprised proteins involved in carbohydrate-, energy-, and amino acid metabolism, and tetrapyrrole biosynthesis. In control plants, five proteins increased, and nine proteins decreased in abundance in the AtMPK6-suppressed genotype compared to that of the WT, reflecting changes in the protein composition of plants that have AtMPK6 constitutively suppressed. Since a number of these proteins are part of the redox response pathway, and loss of AtMPK6 renders Arabidopsis more susceptible to oxidative stress, we propose that AtMPK6 plays a key role in the plant''s overall ability to manage oxidative stress.Key words: Arabidopsis thaliana, AtMPK6, isotope-coded affinity tag (ICAT), ozone, MAPK, signalling     

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.     

拟南芥AtMPK6的信号转导功能和参与发育调控的研究进展

促分裂原活化蛋白激酶（MAPK）级联途径主要MAPKKK、MAPKK和MAPK三个组分构成,彼此逐级磷酸化进而传递细胞信号。这些激酶可以将信息从感应器传递到效应器,并在胞内外信号传递中起多种作用。同时,MAPK级联途径通过相互“交谈”形成复杂的信号传递网络,从而有效地传递各种特异信号。迄今为止,拟南芥AtMPK3、AtMPK4和AtMPK6是研究最多的MAPKs。本文综述AtMPK6参与调控植物对逆境胁迫的响应,以及在生长发育过程中的作用,并介绍AtMPK6与蛋白磷酸酶之间的关系。     

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.     

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.     

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.     

Auxin induces mitogenic activated protein kinase (MAPK) activation in roots of Arabidopsis seedlings

Genome analyses have shown that plants contain gene families encoding various components of mitogen-activated protein kinase (MAPK) signaling pathways. Previous reports have described the involvement of MAPK pathways in stress and pathogen responses of leaves and suspension-cultured cells. Here we show that auxin treatment of Arabidopsis roots transiently induced increases in protein kinase activity with characteristics of mammalian ERK-like MAPKs. The MAPK response we monitored was the result of hormonal action of biologically active auxin, rather than a stress response provoked by auxin-like compounds. Auxin-induced MAPK pathway signaling was distinguished genetically in the Arabidopsis auxin response mutant axr4, in which MAPK activation by auxin, but not by salt stress, was significantly impaired. Perturbation of MAPK signaling in roots using inhibitors of a mammalian MAPKK blocked auxin-activated transgene expression in BA3-GUS seedlings, while potentiating higher than normal levels of MAPK activation in response to auxin. Data presented here indicate that MAPK pathway signaling is positively involved in auxin response, and further suggest that interactions among MAPK signaling pathways in plants influence plant responses to auxin.     

Microbial elicitors induce activation and dual phosphorylation of the Arabidopsis thaliana MAPK 6

Protein kinases related to the family of mitogen-activated kinases (MAPKs) have been established as signal transduction components in a variety of processes in plants. For Arabidopsis thaliana, however, although one of the genetically best studied plant species, biochemical data on activation of mitogen-activated protein kinases are lacking. A. thaliana MAPK 6 (AtMPK6) is the Arabidopsis orthologue of a tobacco MAPK termed salicylate-induced protein kinase, which is activated by general and race-specific elicitors as well as by physical stress. Using a C terminus-specific antibody, we show that AtMPK6 is activated in elicitor-treated cell cultures of A. thaliana. Four different elicitors from bacteria, fungi, and plants lead to a rapid and transient activation of AtMPK6, indicating a conserved signaling pathway. The induction was equally rapid as medium alkalinization, one of the earliest elicitor response observed in cell cultures. A similarly rapid activation of AtMPK6 was observed in elicitor-treated leaf strips, demonstrating that recognition of the elicitors and activation of the MAPK pathway occurs also in intact plants. We demonstrate by in vivo labeling that AtMPK6 is phosphorylated on threonine and tyrosine residues in elicited cells.     

The Arabidopsis MAP kinase kinase 7: A crosstalk point between auxin signaling and defense responses?

Plant-pathogen interaction induces a complex host response that coordinates various signaling pathways through multiple signal molecules. Besides the well-documented signal molecules salicylic acid (SA), ethylene and jasmonic acid, auxin is emerging as an important player in this response. We recently characterized an Arabidopsis activation-tagged mutant, bud1, in which the expression of the MAP kinase kinase 7 (AtMKK7) gene is increased. The bud1 mutant plants accumulate elevated levels of SA and display constitutive pathogenesis-related (PR) gene expression and enhanced resistance to pathogens. Additionally, increased expression of AtMKK7 in the bud1 mutant causes deficiency in polar auxin transport, indicating that AtMKK7 negatively regulates auxin signaling. Based on these results, we hypothesized that AtMKK7 may serve as a crosstalk point between auxin signaling and defense responses. Here we show that increased expression of AtMKK7 in bud1 results in a significant reduction in free auxin (indole-3-acetic acid) levels in the mutant plants. We propose three possible mechanisms to explain how AtMKK7 coordinates the growth hormone auxin and the defense signal molecule SA in the bud1 mutant plants. We suggest that AtMKK7 may play a role in cell death and propose that AtMPK3 and AtMPK6 may function downstream of AtMKK7.Key words: Arabidopsis, MAP kinase kinase 7, auxin signaling, defense responses, crosstalkPathogen invasion of a plant induces multiple physiological changes at the site of infection, including the accumulation of reactive oxygen species, nitric oxide and salicylic acid (SA).^1–6 Jasmonic acid (JA) and ethylene (ET) are also produced in response to pathogen infection.^7–11 Numerous reports have documented that SA, JA and ET work synergistically or antagonistically to fine-tune plant defense responses, based on a multitude of environmental, host and pathogen genetic factors that vary depending on the pathogen-host combinations.^4,12The growth hormone auxin may also play an important role in plant defense responses. Many plant-pathogenic microorganisms have the ability to produce indole-3-acetic acid (IAA),¹³ which is important for the pathogenicity for some pathogens.^14–16 In the Arabidopsis-Xanthomonas campestris pv. campestris (Xcc) compatible interaction, Xcc triggers IAA synthesis in the host plants.17 Exogenous treatment of plants with the auxin analogs, NAA and 2,4-D, leads to disease susceptibility.¹⁸ A flagellin-derived-peptid e-induced microRNA (miRNA) was found to negatively regulate messenger RNAs for the F-box auxin receptors TIR1, AFB2 and AFB3, to repress auxin signaling, resulting in significantly enhanced host resistance.¹⁸ These results suggest that auxin likely functions as a virulence factor to suppress host defense.We previously identified an Arabidopsis activation-tagged mutant bud1 from a transgenic population generated by a sense/antisense RNA expression system.¹⁹ Further characterization indicated that bud1 is a semidominant mutant, in which the expression of the Arabidopsis MAP kinase kinase 7 (AtMKK7) gene is increased.²⁰ The increased expression of AtMKK7 in bud1 causes deficiency in auxin transport, whereas reducing mRNA levels of AtMKK7 by antisense RNA expression leads to enhancement of auxin transport, indicating that AtMKK7 negatively regulates polar auxin transport (PAT).²⁰ Recently, we have shown that the bud1 mutant plants accumulate elevated levels of SA and exhibit constitutive pathogenesis-related (PR) gene expression and enhanced resistance to both the bacterial pathogen Pseudomonas syringae pv. maculicola (Psm) ES4326 and the oomycete pathogen Hyaloperonospora parasitica Noco2.²¹ Reducing mRNA levels of AtMKK7 by antisense RNA expression not only compromises basal resistance but also blocks the induction of systemic acquired resistance (SAR), demonstrating that AtMKK7 is a positive regulator required for both basal resistance and SAR.²¹ Furthermore, we found that the free IAA levels in the bud1 mutant plants were significantly reduced, compared to those in wild-type plants (Fig. 1A). All these results taken together suggest that AtMKK7 may positively regulate SA signaling and negatively regulate auxin signaling.Open in a separate windowFigure 1(A) Free IAA levels in wild type (WT) and bud1 mutant plants. Thirty-day-old soil grown plants were used for free IAA measurement. (B) A schematic representation of three possible mechanisms through which MKK7 regulates host responses after pathogen invasion.Given that SA is a positive regulator of defense responses, whereas auxin is likely a negative regulator of defense responses, we propose three possible mechanisms through which AtMKK7 coordinates the growth hormone auxin and the defense signal molecule SA in the bud1 mutant plants (Fig. 1B): (1) AtMKK7 induces SA accumulation, which suppresses auxin signaling, leading to increased defense responses; (2) AtMKK7 independently induces SA accumulation and suppresses auxin signaling; (3) AtMKK7 suppresses auxin signaling, which relieves the repression of SA signaling by auxin, resulting in SA accumulation.We could test the hypotheses using different approaches. We can examine whether the expression of YUC1, YUC2, YUC4 and YUC6, genes that have been suggested to play essential roles in auxin biosynthesis,²² is altered in the bud1 mutant. We can also analyze the expression of YUC1, YUC2, YUC4 and YUC6, as well as the levels of free IAA in the double mutant bud1sid2 (sid2 is a SA deficient mutant) to test whether IAA biosynthesis is derepressed in the double mutant. Furthermore, polar auxin transport in the bud1sid2 plants should be determined. Finally, we can test whether exogenous application of auxin is able to suppress AtMKK7-induced constitutive defense responses in the bud1 mutant, including elevated levels of SA, constitutive PR gene expression and enhanced resistance to Psm ES4326 and H. parasitica Noco2.AtMKK7 belongs to the Group D of plant MAPKKs.²³ Functions of two other members of this group, LeMKK4 and NbMKK1, have been described.^24,25 LeMKK4 and NbMKK1 are orthologs of AtMKK7 in tomato and Nicotiana benthamiana, respectively. When overexpressed in leaves, wild-type LeMKK4 elicits cell death in both tomato and N. benthamiana.²⁴ Overexpression of wild-type NbMKK1 also causes cell death on N. benthamiana leaves.²⁵ We expected that overexpression of AtMKK7 would also result in cell death. However, neither increased expression of AtMKK7 in the bud1 mutant plants, nor overexpression of wild-type AtMKK7 from the dexamethasone-inducible promoter causes cell death.²¹ This is probably because the expression levels of AtMKK7 in these plants were below the threshold to induce cell death. Consistently, ectopic and constitutive expression of AtMKK7 driven by the cauliflower mosaic virus (CaMV) 35S promoter in wild-type plants leads to lethality of the transgenic plants.²⁰ Therefore, to characterize the function of AtMKK7 in cell death, transgenic plants expressing a constitutively active form of AtMKK7 (AtMKK7^S193A/S199D) from the dexamethasone-inducible promoter will be useful.What MAPK(s) acts downstream of AtMKK7? LeMKK4 directly phosphorylates LeMPK1, LeMPK2 and LeMPK3 in vitro, and activates LeMPK2 and LeMPK3 when expressed in tomato leaves,²⁴ whereas NbMKK1 activates NbSIPK when expressed in N. benthamiana leaves.²⁵ LeMPK2 and LeMPK3 are tomato orthologs of the well-studied tobacco proteins SIPK (salicylic acid-induced protein kinase) and WIPK (wound-induced protein kinase),^26,27 respectively. The Arabidopsis orthologs of SIPK and WIPK are AtMPK6 and AtMPK3, respectively. Based on previous in-gel kinase assay results,²¹ we predict that both AtMPK3 and AtMPK6 may function downstream of AtMKK7. Characterization of double mutants bud1atmpk3 and bud1atmpk6, as well as atmpk3 and atmpk6 mutant plants expressing the constitutively active form of AtMKK7 from the dexamethasone-inducible promoter will shed light on this question.     

Molecular dissection of the response of a rice leucine-rich repeat receptor-like kinase (LRR-RLK) gene to abiotic stresses

Leucine-rich repeat (LRR) receptor-like kinase (RLK) proteins play key roles in a variety of biological pathways. In a previous study, we analyzed the members of the rice LRR-RLK gene family using in silico analysis. A total of 23 LRR-RLK genes were selected based on the expression patterns of a genome-wide dataset of microarrays. The Oryza sativa gamma-ray induced LRR-RLK1 (OsGIRL1) gene was highly induced by gamma irradiation. Therefore, we studied its expression pattern in response to various different abiotic and phytohormone treatments. OsGIRL1 was induced on exposure to abiotic stresses such as salt, osmotic, and heat, salicylic acid (SA), and abscisic acid (ABA), but exhibited downregulation in response to jasmonic acid (JA) treatment. The OsGIRL1 protein was clearly localized at the plasma membrane. The truncated proteins harboring juxtamembrane and kinase domains (or only harboring a kinase domain) exhibited strong autophosphorylation. The biological function of OsGIRL1 was investigated via heterologous overexpression of this gene in Arabidopsis plants subjected to gamma-ray irradiation, salt stress, osmotic stress, and heat stress. A hypersensitive response was observed in response to salt stress and heat stress, whereas a hyposensitive response was observed in response to gamma-ray treatment and osmotic stress. These results provide critical insights into the molecular functions of the rice LRR-RLK genes as receptors of external signals.     

Characterization of mitogen activated protein kinases (MAPKs) in the Curcuma longa expressed sequence tag database

Mitogen activated protein kinase (MAPK) cascades are universal signal transduction modules that play crucial role in plant growth and development as well as biotic and abiotic stress responses. 20 and 17 MAPKs have been characterized in Arabidopsis and rice respectively, which are used for identification of the putative MAPKs in other higher plants. However, no MAPK gene sequences have yet been characterized for asexually reproducing plants. We describe the analysis of MAPK EST sequences from Curcuma longa (an asexually reproducible plant of great medicinal and economic significance). The four Curcuma MAPKs contains all 11 MAPK conserved domains and phosphorylation-activation motif, TEY. Phylogenetic analysis grouped them in the subgroup A and C as identified earlier for Arabidopsis. The Curcuma MAPKs identified showed high sequence homology to rice OsMPK3, OsMPK4 and OsMPK5 suggesting the presence of similar key element in signaling biotic and abiotic stress responses. Although further in vivo and in vitro analysis are required to establish the physiological role of Curcuma MAPKs, this study provides the base for future research on diverse signaling pathways mediated by MAPKs in Curcuma longa as well as other asexually reproducing plants.     

Calmodulin-like protein CML37 is a positive regulator of ABA during drought stress in Arabidopsis

Plants need to adapt to various stress factors originating from the environment. Signal transduction pathways connecting the recognition of environmental cues and the initiation of appropriate downstream responses in plants often involve intracellular Ca²⁺ concentration changes. These changes must be deciphered into specific cellular signals. Calmodulin-like proteins, CMLs, act as Ca²⁺ sensors in plants and are known to be involved in various stress reactions. Here, we show that in Arabidopsis 2 different CMLs, AtCML37 and AtCML42 are antagonistically involved in drought stress response. Whereas a CML37 knock-out line, cml37, was highly susceptible to drought stress, CML42 knockout line, cml42, showed no obvious effect compared to wild type (WT) plants. Accordingly, the analysis of the phytohormone abscisic acid (ABA) revealed a significant reduction of ABA upon drought stress in cml37 plants, while in cml42 plants an increase of ABA was detected. Summarizing, our results show that both CML37 and CML42 are involved in drought stress response but show antagonistic effects.