Characterization of PsMPK2, the first C1 subgroup MAP kinase from pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

Mitogen-activated protein kinase (MAPK) cascades play a key role in plant growth and development as well as in biotic and abiotic stress responses. They are classified according to their sequence homology into four major groups (A–D). A large amount of information about MAPKs in groups A and B is available but few data of the C group have been reported. In this study, a C1 subgroup MAP kinase cDNA, PsMPK2, was isolated from Pisum sativum. PsMPK2 is expressed in vegetative (root and leaf) and reproductive (stamen, pistil and fruit) organs. Expression of PsMPK2 in Arabidopsis thaliana shows that mechanical injury and other stress signals as abscisic acid, jasmonic acid and hydrogen peroxide increase its kinase activity, extending previous results indicating that C1 subgroup MAPKs may be involved in the response to stress.     

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.     

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 PP2C-type phosphatase AP2C1, which negatively regulates MPK4 and MPK6, modulates innate immunity, jasmonic acid, and ethylene levels in Arabidopsis

Wound signaling pathways in plants are mediated by mitogen-activated protein kinases (MAPKs) and stress hormones, such as ethylene and jasmonates. In Arabidopsis thaliana, the transmission of wound signals by MAPKs has been the subject of detailed investigations; however, the involvement of specific phosphatases in wound signaling is not known. Here, we show that AP2C1, an Arabidopsis Ser/Thr phosphatase of type 2C, is a novel stress signal regulator that inactivates the stress-responsive MAPKs MPK4 and MPK6. Mutant ap2c1 plants produce significantly higher amounts of jasmonate upon wounding and are more resistant to phytophagous mites (Tetranychus urticae). Plants with increased AP2C1 levels display lower wound activation of MAPKs, reduced ethylene production, and compromised innate immunity against the necrotrophic pathogen Botrytis cinerea. Our results demonstrate a key role for the AP2C1 phosphatase in regulating stress hormone levels, defense responses, and MAPK activities in Arabidopsis and provide evidence that the activity of AP2C1 might control the plant's response to B. cinerea.     

A mitogen-activated protein kinase NtMPK4 activated by SIPKK is required for jasmonic acid signaling and involved in ozone tolerance via stomatal movement in tobacco

The mitogen-activated protein kinase (MAPK) cascade is involved in responses to biotic and abiotic stress in plants. In this study, we isolated a new MAPK, NtMPK4, which is a tobacco homolog of Arabidopsis MPK4 (AtMPK4). NtMPK4 was activated by wounding along with two other wound-responsive tobacco MAPKs, WIPK and SIPK. We found that NtMPK4 was activated by salicylic acid-induced protein kinase kinase (SIPKK), which has been isolated as an SIPK-interacting MAPK kinase. In NtMPK4 activity-suppressed tobacco, wound-induced expression of jasmonic acid (JA)-responsive genes was inhibited. NtMPK4-silenced plants showed enhanced sensitivity to ozone. Inversely, transgenic tobacco plants, in which SIPKK or the constitutively active type SIPKK(EE) was overexpressed, exhibited greater responsiveness to wounding with enhanced resistance to ozone. We further found that NtMPK4 was expressed preferentially in epidermis, and the enhanced sensitivity to ozone in NtMPK4-silenced plants was caused by an abnormal regulation of stomatal closure in an ABA-independent manner. These results suggest that NtMPK4 is involved in JA signaling and in stomatal movement.     

An Arabidopsis protein phosphorylated in response to microbial elicitation, AtPHOS32, is a substrate of MAP kinases 3 and 6

Although mitogen-activated protein kinases (MAPKs) have been shown to be activated by a wide range of biotic and abiotic stimuli in diverse plant species, few in vivo substrates for these kinases have been identified. While studying proteins that are differentially phosphorylated upon treatment of Arabidopsis suspension cultures with the general bacterial elicitor peptide flagellin-22 (flg22), we identified two proteins with endogenous nickel binding properties that become phosphorylated after flg22 elicitation. These highly related proteins, AtPHOS32 and AtPHOS34, show similarity to bacterial universal stress protein A. We identified one of the phosphorylation sites on AtPHOS32 by nanoelectrospray ionization tandem mass spectrometry. Phosphorylation in a phosphoSer-Pro motif indicated that this protein may be a substrate of MAPKs. Using in vitro kinase assays, we confirmed that AtPHOS32 is a substrate of both AtMPK3 and AtMPK6. Specificity of phosphorylation was demonstrated by site-directed mutagenesis of the first phosphorylation site. In addition, immunosubtraction of both MAPKs from protein extracts removed detectable kinase activity toward AtPHOS32, indicating that the two MAPKs were the predominate kinases recognizing the motif in this protein. Finally, the target phosphorylation site in AtPHOS32 is conserved in AtPHOS34 and among apparent orthologues from many plant species, indicating that phosphorylation of these proteins by AtMPK3 and AtMPK6 orthologues has been conserved throughout evolution.     

Identification of a dual-specificity protein phosphatase that inactivates a MAP kinase from Arabidopsis

Mitogen-activated protein kinases (MAPKs) play a key role in plant responses to stress and pathogens. Activation and inactivation of MAPKs involve phosphorylation and dephosphorylation on both threonine and tyrosine residues in the kinase domain. Here we report the identification of an Arabidopsis gene encoding a dual-specificity protein phosphatase capable of hydrolysing both phosphoserine/threonine and phosphotyrosine in protein substrates. This enzyme, designated AtDsPTP1 (Arabidopsis thaliana dual-specificity protein tyrosine phosphatase), dephosphorylated and inactivated AtMPK4, a MAPK member from the same plant. Replacement of a highly conserved cysteine by serine abolished phosphatase activity of AtDsPTP1, indicating a conserved catalytic mechanism of dual-specificity protein phosphatases from all eukaryotes.     

Reassessing the role of phospholipase D in the Arabidopsis wounding response

Plants respond to wounding by means of a multitude of reactions, with the purpose of stifling herbivore assault. Phospholipase D (PLD) has previously been implicated in the wounding response. Arabidopsis ( Arabidopsis thaliana ) AtPLD α 1 has been proposed to be activated in intact cells, and the phosphatidic acid (PA) it produces to serve as a precursor for jasmonic acid (JA) synthesis and to be required for wounding-induced gene expression. Independently, PLD activity has been reported to have a bearing on wounding-induced MAPK activation. However, which PLD isoforms are activated, where this activity takes place (in the wounded or non-wounded cells) and what exactly the consequences are is a question that has not been comprehensively addressed. Here, we show that PLD activity during the wounding response is restricted to the ruptured cells using ³²P_i-labelled phospholipid analyses of Arabidopsis pld knock-out mutants and PLD -silenced tomato cell-suspension cultures. pldα1 knock-out lines have reduced wounding-induced PA production, and the remainder is completely eliminated in a pldα1 / δ double knock-out line. Surprisingly, wounding-induced protein kinase activation, AtLOX2 gene expression and JA biosynthesis were not affected in these knock-out lines. Moreover, larvae of the Cabbage White butterfly ( Pieris rapae ) grew equally well on wild-type and the pld knock-out mutants.     

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.     

A calmodulin-binding mitogen-activated protein kinase phosphatase is induced by wounding and regulates the activities of stress-related mitogen-activated protein kinases in rice

The mitogen-activated protein kinase (MAPK) phosphatases (MKPs) are negative regulators of MAPKs. In dicotyledons such as Arabidopsis and tobacco, MKPs have been shown to play pivotal roles in abiotic stress responses, hormone responses and microtubule organization. However, little is known about the role of MKPs in monocotyledons such as rice. Database searches identified five putative MKPs in rice. We investigated their expression in response to wounding, and found that the expression of OsMKP1 is rapidly induced by wounding. In this study, we functionally characterized the involvement of OsMKP1 in wound responses. The deduced amino acid sequence of OsMKP1 shows strong similarity to Arabidopsis AtMKP1 and tobacco NtMKP1. Moreover, OsMKP1 bound calmodulin in a manner similar to NtMKP1. To determine the biological function of OsMKP1, we obtained osmkp1, a loss-of-function mutant, in which retrotransposon Tos17 was inserted in the second exon of OsMKP1. Unlike the Arabidopsis atmkp1 loss-of-function mutant, which shows no abnormal phenotype without stimuli, osmkp1 showed a semi-dwarf phenotype. Exogenous supply of neither gibberellin nor brassinosteroid complemented the semi-dwarf phenotype of osmkp1. Activities of two stress-responsive MAPKs, OsMPK3 and OsMPK6, in osmkp1 were higher than those in the wild type both before and after wounding. Microarray analysis identified 13 up-regulated and eight down-regulated genes in osmkp1. Among the up-regulated genes, the expression of five genes showed clear responses to wounding, indicating that wound responses are constitutively activated in osmkp1. These results suggest that OsMKP1 is involved in the negative regulation of rice wound responses.     

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.     

Arabidopsis MAP kinase phosphatase 1 is phosphorylated and activated by its substrate AtMPK6

Arabidopsis MAP kinase phosphatase 1 (AtMKP1) is a member of the mitogen-activated protein kinase (MPK) phosphatase family, which negatively regulates AtMPKs. We have previously shown that AtMKP1 is regulated by calmodulin (CaM). Here, we examined the phosphorylation of AtMKP1 by its substrate AtMPK6. Intriguingly, AtMKP1 was phosphorylated by AtMPK6, one of AtMKP1 substrates. Four phosphorylation sites were identified by phosphoamino acid analysis, TiO(2) chromatography and mass spectrometric analysis. Site-directed mutation of these residues in AtMKP1 abolished the phosphorylation by AtMPK6. In addition, AtMKP1 interacted with AtMPK6 as demonstrated by the yeast two-hybrid system. Finally, the phosphatase activity of AtMKP1 increased approximately twofold following phosphorylation by AtMPK6. By in-gel kinase assays, we showed that AtMKP1 could be rapidly phosphorylated by AtMPK6 in plants. Our results suggest that the catalytic activity of AtMKP1 in plants can be regulated not only by Ca(2+)/CaM, but also by its physiological substrate, AtMPK6.     

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     

Novel rice MAP kinases OsMSRMK3 and OsWJUMK1 involved in encountering diverse environmental stresses and developmental regulation

We report isolation of two novel rice (Oryza sativa L.) mitogen-activated protein kinases (MAPKs), OsMSRMK3 (multiple stress responsive) and OsWJUMK1 (wound- and JA-uninducible) that most likely exist as single copy genes in its genome. OsMSRMK3 and OsWJUMK1 encode 369 and 569 amino acid polypeptides having the MAPK family signature and phosphorylation activation motifs TEY and TDY, respectively. Steady state mRNA analyses of these MAPKs with constitutive expression in leaves of two-week-old seedlings revealed that OsMSRMK3 was up-regulated upon wounding (by cut), jasmonic acid (JA), salicylic acid (SA), ethylene, abscisic acid, hydrogen peroxide (H(2)O(2)), protein phosphatase inhibitors, chitosan, high salt/sugar, and heavy metals, whereas OsWJUMK1 not induced by either wounding, JA or SA, showed up-regulation only by H(2)O(2), heavy metals, and cold stress (12 degrees C). Moreover, these MAPKs were developmentally regulated. These results strongly suggest a role for OsMSRMK3 and OsWJUMK1 in both stress-signalling pathways and development in rice.