Arabidopsis Mitogen-activated Protein Kinase Research Articles

Subfamily C7 Raf-like kinases MRK1, RAF26, and RAF39 regulate immune homeostasis and stomatal opening in Arabidopsis thaliana.

The calcium-dependent protein kinase CPK28 regulates several stress pathways in multiple plant species. Here, we aimed to discover CPK28-associated proteins in Arabidopsis thaliana. We used affinity-based proteomics and identified several potential CPK28 binding partners, including the C7 Raf-like kinases MRK1, RAF26, and RAF39. We used biochemistry, genetics, and physiological assays to gain insight into their function. We define redundant roles for these kinases in stomatal opening, immune-triggered reactive oxygen species (ROS) production, and resistance to a bacterial pathogen. We report that CPK28 associates with and trans-phosphorylates RAF26 and RAF39, and that MRK1, RAF26, and RAF39 are active kinases that localize to endomembranes. Although Raf-like kinases share some features with mitogen-activated protein kinase kinase kinases (MKKKs), we found that MRK1, RAF26, and RAF39 are unable to trans-phosphorylate any of the 10 Arabidopsis mitogen-activated protein kinase kinases (MKKs). Overall, our study suggests that C7 Raf-like kinases associate with and are phosphorylated by CPK28, function redundantly in stomatal opening and immunity, and possess substrate specificities distinct from canonical MKKKs.

Multilayered synergistic regulation of phytoalexin biosynthesis by ethylene, jasmonate, and MAPK signaling pathways in Arabidopsis.

Camalexin, an indolic antimicrobial metabolite, is the major phytoalexin in Arabidopsis thaliana, and plays a crucial role in pathogen resistance. Our previous studies revealed that the Arabidopsis mitogen-activated protein kinases MPK3 and MPK6 positively regulate pathogen-induced camalexin biosynthesis via phosphoactivating the transcription factor WRKY33. Here, we report that the ethylene and jasmonate (JA) pathways act synergistically with the MPK3/MPK6-WRKY33 module at multiple levels to induce camalexin biosynthesis in Arabidopsis upon pathogen infection. The ETHYLENE RESPONSE FACTOR1 (ERF1) transcription factor integrates the ethylene and JA pathways to induce camalexin biosynthesis via directly upregulating camalexin biosynthetic genes. ERF1 also interacts with and depends on WRKY33 to upregulate camalexin biosynthetic genes, indicating that ERF1 and WRKY33 form transcriptional complexes to cooperatively activate camalexin biosynthetic genes, thereby mediating the synergy of ethylene/JA and MPK3/MPK6 signaling pathways to induce camalexin biosynthesis. Moreover, as an integrator of the ethylene and JA pathways, ERF1 also acts as a substrate of MPK3/MPK6, which phosphorylate ERF1 to increase its transactivation activity and therefore further cooperate with the ethylene/JA pathways to induce camalexin biosynthesis. Taken together, our data reveal the multilayered synergistic regulation of camalexin biosynthesis by ethylene, JA, and MPK3/MPK6 signaling pathways via ERF1 and WRKY33 transcription factors in Arabidopsis.

Regulation of Arabidopsis Matrix Metalloproteinases by Mitogen-Activated Protein Kinases and Their Function in Leaf Senescence

Leaf senescence is a developmentally programmed cell death process that is influenced by a variety of endogenous signals and environmental factors. Here, we report that MPK3 and MPK6, two Arabidopsis mitogen-activated protein kinases (MAPKs or MPKs), and their two upstream MAPK kinases (MAPKKs or MKKs), MKK4 and MKK5, are key regulators of leaf senescence. Weak induction of constitutively active MAPKKs driven by steroid-inducible promoter, which activates endogenous MPK3 and MPK6, induces leaf senescence. This gain-of-function phenotype requires functional endogenous MPK3 and MPK6. Furthermore, loss of function of both MKK4 and MKK5 delays leaf senescence. Expression profiling leads to the identification of matrix metalloproteinases (MMPs), a family of zinc- and calcium-dependent endopeptidases, as the downstream target genes of MPK3/MPK6 cascade. MPK3/MPK6 activation-triggered leaf senescence is associated with rapid and strong induction of At3-MMP and At2-MMP. Expression of Arabidopsis MMP genes is strongly induced during leaf senescence, qualifying them as senescence-associated genes (SAGs). In addition, either constitutive or inducible overexpression of At3-MMP is sufficient to trigger leaf senescence. Based on these findings, we conclude that MPK3/MPK6 MAPK cascade and MMP target genes further downstream are involved in regulating leaf senescence in Arabidopsis.

Simultaneous mutations in SMN1 and SUMM2 fully suppress the dwarf and autoimmune phenotypes of Arabidopsis mpk4 mutant

ABSTRACT Disruption of the Arabidopsis mitogen-activated protein kinase pathway, MEKK1–MKK1/MKK2–MPK4 (hereafter designated as MEKK1 pathway), leads to the activation of distinct NLRs (nucleotide-binding and leucine-rich repeat receptors), TNL (TIR-type NLR) SMN1, and CNL (CC-type NLR) SUMM2, resulting in dwarf and autoimmune phenotypes. Unlike mekk1 and mkk1mkk2 mutants, the dwarf and autoimmune phenotypes of mpk4 are only partially suppressed by the summ2 mutation, suggesting a significant contribution of SMN1 to the mpk4 phenotypes. However, full suppression of mpk4 by the smn1summ2 double mutation remains to be elucidated. To address this key question, we generated a mpk4smn1summ2 triple mutant and analyzed the dwarf and constitutive cell death phenotypes. The mpk4smn1summ2 triple mutant showed restoration of plant size with no detectable cell death, indicating full suppression of the dwarf and autoimmune phenotypes. These results suggest that SMN1 and SUMM2 constitute a robust surveillance system for the MEKK1 pathway against pathogen infection.

Dual control of MAPK activities by AP2C1 and MKP1 MAPK phosphatases regulates defence responses in Arabidopsis.

Mitogen-activated protein kinase (MAPK) cascades transmit environmental signals and induce stress and defence responses in plants. These signalling cascades are negatively controlled by specific Ser/Thr protein phosphatases of the type 2C (PP2C) and dual-specificity phosphatase (DSP) families that inactivate stress-induced MAPKs; however, the interplay between phosphatases of these different types has remained unknown. This work reveals that different Arabidopsis MAPK phosphatases, the PP2C-type AP2C1 and the DSP-type MKP1, exhibit both specific and overlapping functions in plant stress responses. Each single mutant, ap2c1 and mkp1, and the ap2c1 mkp1 double mutant displayed enhanced stress-induced activation of the MAPKs MPK3, MPK4, and MPK6, as well as induction of a set of transcription factors. Moreover, ap2c1 mkp1 double mutants showed an autoimmune-like response, associated with increased levels of the stress hormones salicylic acid and ethylene, and of the phytoalexin camalexin. This phenotype was reduced in the ap2c1 mkp1 mpk3 and ap2c1 mkp1 mpk6 triple mutants, suggesting that the autoimmune-like response is due to MAPK misregulation. We conclude that the evolutionarily distant MAPK phosphatases AP2C1 and MKP1 contribute crucially to the tight control of MAPK activities, ensuring appropriately balanced stress signalling and suppression of autoimmune-like responses during plant growth and development.

The mitogen-activated protein kinase kinase MKK2 positively regulates constitutive cold resistance in the potato

Cold stress is a detrimental environmental factor affecting plant growth and development. The mitogen-activated protein kinase (MAPK) cascade pathway mediates various signalling pathways associated with abiotic stress tolerance in plants. In this research, 15 MAPKs, 5 MAPKKs and 83 MAPKKKs in the potato genome were identified by comparative genomics analysis and named following the Arabidopsis MAPK nomenclature model. The differentially expressed genes in response to cold stress between S. acaule and S. tuberosum were enriched in the MAPK cascade pathway. Quantitative real-time PCR (qRT-PCR) results revealed that the expression of SaMKK2 and SaMAPK16 particularly increased in the cold-tolerant S. acaule. Additionally, the promoters of StMKK2 and SaMKK2 differ largely in cold responsive motifs. Compared with wild-type E3, transgenic lines overexpressing SaMKK2 exhibited dramatic improvement of constitutive cold resistance in the potato, indicating that the function of MKK2 is species-dependent. However, SaMAPK16 overexpression had no effects on either cold resistance without or with cold acclimation. Furthermore, the transcriptome and qRT-PCR data showed that overexpression of SaMKK2 led to a significant increase in the expression of CBF1/2/3, OPR2 and SLD2 after cold treatment for two hours compared with wild-type under cold stress. The results of yeast two-hybrid and bimolecular fluorescence complementation experiments together suggested that SaMKK2 interacted with SaMAPK4/7 and that the interaction occurred in the cytoplasm. Given these results, we concluded that the interaction between SaMKK2 and SaMAPK4/7 may positively promote the expression of CBFs, OPR2 and SLD2, which eventually increases the constitutive cold resistance of potato.

MKK4/5-MPK3/6 Cascade Regulates Agrobacterium-Mediated Transformation by Modulating Plant Immunity in Arabidopsis.

Agrobacterium tumefaciens is a specialized plant pathogen that causes crown gall disease and is commonly used for Agrobacterium-mediated transformation. As a pathogen, Agrobacterium triggers plant immunity, which affects transformation. However, the signaling components and pathways in plant immunity to Agrobacterium remain elusive. We demonstrate that two Arabidopsis mitogen-activated protein kinase kinases (MAPKKs) MKK4/MKK5 and their downstream mitogen-activated protein kinases (MAPKs) MPK3/MPK6 play major roles in both Agrobacterium-triggered immunity and Agrobacterium-mediated transformation. Agrobacteria induce MPK3/MPK6 activity and the expression of plant defense response genes at a very early stage. This process is dependent on the MKK4/MKK5 function. The loss of the function of MKK4 and MKK5 or their downstream MPK3 and MPK6 abolishes plant immunity to agrobacteria and increases transformation frequency, whereas the activation of MKK4 and MKK5 enhances plant immunity and represses transformation. Global transcriptome analysis indicates that agrobacteria induce various plant defense pathways, including reactive oxygen species (ROS) production, ethylene (ET), and salicylic acid- (SA-) mediated defense responses, and that MKK4/MKK5 is essential for the induction of these pathways. The activation of MKK4 and MKK5 promotes ROS production and cell death during agrobacteria infection. Based on these results, we propose that the MKK4/5-MPK3/6 cascade is an essential signaling pathway regulating Agrobacterium-mediated transformation through the modulation of Agrobacterium-triggered plant immunity.

Phosphatidic acid promotes the activation and plasma membrane localization of MKK7 and MKK9 in response to salt stress

Phosphatidic acid (PA) is a lipid secondary messenger involved in intracellular signaling in eukaryotes. It has been confirmed that PA mediates salt stress signaling by promoting activation of Mitogen-activated Protein Kinase 6 (MPK6) which phosphorylates Na+/H+ antiporter SOS1. However, the MPK6-upstream kinases and their relationship to PA remain unclear. Here, we found that, among the six tested Arabidopsis Mitogen-activated Protein Kinase Kinases (MKKs), PA specifically bound to MKK7 and MKK9 which phosphorylate MPK6, and promoted the activation of MKK7/MKK9. Based on phenotypic and physiological analyses, we found that MKK7 and MKK9 positively regulate Arabidopsis salt tolerance and are functionally redundant. NaCl treatment can induce significant increase in MKK7/MKK9 activities, and this depends, in part, on the Phospholipase Dα1 (PLDα1). MKK7 and MKK9 also mediate the NaCl-induced activation of MPK6. Furthermore, PA or NaCl treatment could induce translocation of MKK7/MKK9 to the plasma membrane, whereas this translocation disappeared in pldα1. These results indicate that PA binds to MKK7 and MKK9, increases their kinase activity and plasma membrane localization during Arabidopsis response to salt stress. Together with the PA-MPK6-SOS1 pathway identified previously, this mechanism may maximize the signal transduction efficiency, providing novel insights into the link between lipid signaling and MAPK cascade.

A Förster resonance energy transfer sensor for live-cell imaging of mitogen-activated protein kinase activity in Arabidopsis.

The catalytic activity of mitogen-activated protein kinases (MAPKs) is dynamically modified in plants. Since MAPKs have been shown to play important roles in a wide range of signaling pathways, the ability to monitor MAPK activity in living plant cells would be valuable. Here, we report the development of a genetically encoded MAPK activity sensor for use in Arabidopsis thaliana. The sensor is composed of yellow and blue fluorescent proteins, a phosphopeptide binding domain, a MAPK substrate domain and a flexible linker. Using invitro testing, we demonstrated that phosphorylation causes an increase in the Förster resonance energy transfer (FRET) efficiency of the sensor. The FRET efficiency can therefore serve as a readout of kinase activity. We also produced transgenic Arabidopsis lines expressing this sensor of MAPK activity (SOMA) and performed live-cell imaging experiments using detached cotyledons. Treatment with NaCl, the synthetic flagellin peptide flg22 and chitin all led to rapid gains in FRET efficiency. Control lines expressing a version of SOMA in which the phosphosite was mutated to an alanine did not show any substantial changes in FRET. We also expressed the sensor in a conditional loss-of-function double-mutant line for the Arabidopsis MAPK genes MPK3 and MPK6. These experiments demonstrated that MPK3/6 are necessary for the NaCl-induced FRET gain of the sensor, while other MAPKs are probably contributing to the chitin and flg22-induced increases in FRET. Taken together, our results suggest that SOMA is able to dynamically report MAPK activity in living plant cells.

Quantitative Phosphoproteomic Analysis Reveals Shared and Specific Targets of Arabidopsis Mitogen-Activated Protein Kinases (MAPKs) MPK3, MPK4, and MPK6

In Arabidopsis, mitogen-activated protein kinases MPK3, MPK4, and MPK6 constitute essential relays for a variety of functions including cell division, development and innate immunity. Although some substrates of MPK3, MPK4 and MPK6 have been identified, the picture is still far from complete. To identify substrates of these MAPKs likely involved in cell division, growth and development we compared the phosphoproteomes of wild-type and mpk3, mpk4, and mpk6. To study the function of these MAPKs in innate immunity, we analyzed their phosphoproteomes following microbe-associated molecular pattern (MAMP) treatment. Partially overlapping substrates were retrieved for all three MAPKs, showing target specificity to one, two or all three MAPKs in different biological processes. More precisely, our results illustrate the fact that the entity to be defined as a specific or a shared substrate for MAPKs is not a phosphoprotein but a particular (S/T)P phosphorylation site in a given protein. One hundred fifty-two peptides were identified to be differentially phosphorylated in response to MAMP treatment and/or when compared between genotypes and 70 of them could be classified as putative MAPK targets. Biochemical analysis of a number of putative MAPK substrates by phosphorylation and interaction assays confirmed the global phosphoproteome approach. Our study also expands the set of MAPK substrates to involve other protein kinases, including calcium-dependent (CDPK) and sugar nonfermenting (SnRK) protein kinases.

Maternal control of embryogenesis by MPK6 and its upstream MKK4/MKK5 in Arabidopsis.

In flowering plants, developing embryos reside in maternal sporophytes. It is known that maternal generation influences the development of next-generation embryos; however, little is known about the signaling components in the process. Previously, we demonstrated that Arabidopsis mitogen-activated protein kinase6 (MPK6) and MPK3 play critical roles in plant reproduction. In addition, we noticed that a large fraction of seeds from mpk6 single-mutant plants showed a wrinkled seed coat or a burst-out embryo phenotype. Here, we report that these seed phenotypes can be traced back to defective embryogenesis. The defective embryos have shorter suspensors and reduced growth along the longitudinal axis. Furthermore, the cotyledons fail to bend over to progress to the bent-cotyledon stage. As a result of the uneven circumference along the axis, the seed coat wrinkles to develop raisin-like morphology after dehydration. In more severe cases, the embryo can be pushed out from the micropylar end, resulting in the burst-out embryo seed phenotype. Genetic analyses demonstrated that the defective embryogenesis of the mpk6 mutant is a maternal effect. Heterozygous or homozygous mpk6 embryos have defects only in mpk6 homozygous maternal plants, but not in wild-type or heterozygous maternal plants. The loss of function of MKK4/MKK5 also results in the same phenotypes, suggesting that MKK4/MKK5 might act upstream of MPK6 in this pathway. The maternal-mediated embryo defects are associated with changes in auxin activity maxima and PIN localization. In summary, this research demonstrates that the Arabidopsis MKK4/MKK5-MPK6 cascade is an important player in the maternal control of embryogenesis.

Substrate thiophosphorylation by Arabidopsis mitogen-activated protein kinases.

BackgroundMitogen-activated protein kinase (MPK) cascades are important to cellular signaling in eukaryotes. They regulate growth, development and the response to environmental challenges. MPK cascades function via reversible phosphorylation of cascade components, MEKK, MEK, and MPK, but also by MPK substrate phosphorylation. Using mass spectrometry, we previously identified many in vivo MPK3 and MPK6 substrates in Arabidopsis thaliana, and we disclosed their phosphorylation sites.ResultsWe verified phosphorylation of several of our previously identified MPK3/6 substrates using a nonradioactive in vitro labeling assay. We engineered MPK3, MPK4, and MPK6 to accept bio-orthogonal ATPγS analogs for thiophosphorylating their appropriate substrate proteins. Subsequent alkylation of the thiophosphorylated amino acid residue(s) allows immunodetection using thiophosphate ester-specific antibodies. Site-directed mutagenesis of amino acids confirmed the protein substrates’ site-specific phosphorylation by MPK3 and MPK6. A combined assay with MPK3, MPK6, and MPK4 revealed substrate specificity of the individual kinases.ConclusionOur work demonstrates that the in vitro-labeling assay represents an effective, specific and highly sensitive test for determining kinase-substrate relationships.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0731-6) contains supplementary material, which is available to authorized users.

Genome-wide identification and transcriptional expression analysis of mitogen-activated protein kinase and mitogen-activated protein kinase kinase genes in Capsicum annuum.

The tripartite mitogen-activated protein kinase (MAPK) signaling cascades have been implicated in plant growth, development, and environment adaptation, but a comprehensive understanding of MAPK signaling at genome-wide level is limited in Capsicum annuum. Herein, genome-wide identification and transcriptional expression analysis of MAPK and MAPK kinase (MAPKK) were performed in pepper. A total of 19 pepper MAPK (CaMAPKs) genes and five MAPKK (CaMAPKKs) genes were identified. Phylogenetic analysis indicated that CaMAPKs and CaMAPKKs could be classified into four groups and each group contains similar exon-intron structures. However, significant divergences were also found. Notably, five members of the pepper MAPKK family were much less conserved than those found in Arabidopsis, and 9 Arabidopsis MAPKs did not have orthologs in pepper. Additionally, 7 MAPKs in Arabidopsis had either two or three orthologs in the pepper genome, and six pepper MAPKs and one MAPKK differing in sequence were found in three pepper varieties. Quantitative real-time RT-PCR analysis showed that the majority of MAPK and MAPKK genes were ubiquitously expressed and transcriptionally modified in pepper leaves after treatments with heat, salt, and Ralstonia solanacearum inoculation as well as exogenously applied salicylic acid, methyl jasmonate, ethephon, and abscisic acid. The MAPKK-MAPK interactome was tested by yeast two-hybrid assay, the results showed that one MAPKK might interact with multiple MAPKs, one MAPK might also interact with more than one MAPKKs, constituting MAPK signaling networks which may collaborate in transmitting upstream signals into appropriate downstream cellular responses and processes. These results will facilitate future functional characterization of MAPK cascades in pepper.

Sclareol induces plant resistance to root-knot nematode partially through ethylene-dependent enhancement of lignin accumulation.

The root-knot nematode (RKN) is one of the most devastating parasitic nematodes of plants. Although some secondary metabolites released by the host plant play roles as defense substances against parasitic nematodes, the mechanism underlying the induction of such defense responses is not fully understood. We found that sclareol, a natural diterpene known as an antimicrobial and defense-related molecule, inhibited RKN penetration of tomato and Arabidopsis roots. Sclareol induced genes related to ethylene (ET) biosynthesis and signaling and phenylpropanoid metabolism in Arabidopsis roots. In roots of ein2-1, an ET-insensitive mutant line, both sclareol-induced inhibition of RKN penetration and sclareol-induced enhancement of lignin accumulation were abolished. A mutant defective in lignin accumulation did not exhibit such inhibition. Sclareol also activated MPK3 and MPK6, Arabidopsis mitogen-activated protein kinases whose activation is required for triggering ET biosynthesis. Sclareol-induced inhibition of RKN penetration was exhibited by mutants of neither MPK3 nor MPK6. Treatment with a biosynthetic precursor of ET was insufficient compared with sclareol treatment to inhibit RKN penetration, suggesting the existence of an ET-independent signaling pathway leading to RKN resistance. These results suggested that sclareol induced resistance to RKN penetration partially through ET-dependent accumulation of lignin in roots.

Timing Is Everything: Highly Specific and Transient Expression of a MAP Kinase Determines Auxin-Induced Leaf Venation Patterns in Arabidopsis

Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules present in all eukaryotes. In plants, MAPK cascades were shown to regulate cell division, developmental processes, stress responses, and hormone pathways. The subgroup A of Arabidopsis MAPKs consists of AtMPK3, AtMPK6, and AtMPK10. AtMPK3 and AtMPK6 are activated by their upstream MAP kinase kinases (MKKs) AtMKK4 and AtMKK5 in response to biotic and abiotic stress. In addition, they were identified as key regulators of stomatal development and patterning. AtMPK10 has long been considered as a pseudo-gene, derived from a gene duplication of AtMPK6. Here we show that AtMPK10 is expressed highly but very transiently in seedlings and at sites of local auxin maxima leaves. MPK10 encodes a functional kinase and interacts with the upstream MAP kinase kinase (MAPKK) AtMKK2. mpk10 mutants are delayed in flowering in long-day conditions and in continuous light. Moreover, cotyledons of mpk10 and mkk2 mutants have reduced vein complexity, which can be reversed by inhibiting polar auxin transport (PAT). Auxin does not affect AtMPK10 expression while treatment with the PAT inhibitor HFCA extends the expression in leaves and reverses the mpk10 mutant phenotype. These results suggest that the AtMKK2-AtMPK10 MAPK module regulates venation complexity by altering PAT efficiency.

The Salmonella effector protein SpvC, a phosphothreonine lyase is functional in plant cells.

Salmonella is one of the most prominent causes of food poisoning and growing evidence indicates that contaminated fruits and vegetables are an increasing concern for human health. Successful infection demands the suppression of the host immune system, which is often achieved via injection of bacterial effector proteins into host cells. In this report we present the function of Salmonella effector protein in plant cell, supporting the new concept of trans-kingdom competence of this bacterium. We screened a range of Salmonella Typhimurium effector proteins for interference with plant immunity. Among these, the phosphothreonine lyase SpvC attenuated the induction of immunity-related genes when present in plant cells. Using in vitro and in vivo systems we show that this effector protein interacts with and dephosphorylates activated Arabidopsis Mitogen-activated Protein Kinase 6 (MPK6), thereby inhibiting defense signaling. Moreover, the requirement of Salmonella SpvC was shown by the decreased proliferation of the ΔspvC mutant in Arabidopsis plants. These results suggest that some Salmonella effector proteins could have a conserved function during proliferation in different hosts. The fact that Salmonella and other Enterobacteriaceae use plants as hosts strongly suggests that plants represent a much larger reservoir for animal pathogens than so far estimated.

Bimolecular fluorescent complementation (BiFC) by MAP kinases and MAPK phosphatases.

The adaptation of plants to the environment is a key property for survival. Adaptation responses to environmental cues are generated in cells by signaling initiated from cell receptors. Signal transduction is based on protein phosphorylation that is employed in mitogen-activated protein kinase (MAPK) cascades to integrate signals from receptors to cellular responses. MAPK activity is determined by phosphorylation of amino acid residues within the kinase activation loop and their dephosphorylation by phosphatases is essential to control signal duration and intensity.Monitoring protein-protein interactions (PPIs) of MAPKs with MAPK phosphatases in vivo provides valuable information about specificity and intracellular localization of the protein complex. Here, we report studying PPIs between Arabidopsis MAPKs and PP2C-type MAPK phosphatases using bimolecular fluorescent complementation (BiFC) in suspension cell protoplasts. The interactions of the MAPKs MPK3, MKP4 and MPK6 with the phosphatases AP2C1 and AP2C3 have been tested.

Transient expression in Arabidopsis leaf mesophyll protoplast system for cell-based functional analysis of MAPK cascades signaling.

Mitogen-Activated Protein Kinase (MAPK) cascade is one of the main signaling components mediating abiotic and biotic stress and hormone information in plants. Plant MAPK study has been impeded with a genetic approach using a long-term phenotypic analysis in spite of the transient nature of the protein kinase signaling. Arabidopsis leaf mesophyll protoplasts provide a versatile resource for diverse cell-based assays to acquire immediate molecular and biochemical responses with transient expression of MAPK cascade components of interests. Thus, it is an attractive tool for a high-throughput functional analysis of Arabidopsis MAPK cascade signaling. However, transient expression in Arabidopsis mesophyll protoplast (TEAMP) system requires mastered skills for protoplast preparation and handling to achieve steady and stable data. Here, we have described two analytical methods for MAPK cascade signaling using TEAMP system.

Fluorescent protein tagging of Arabidopsis MAPKs for in vivo localization studies.

Mitogen-activated protein kinases (MAPK) are key regulatory elements in many processes. They are highly conserved throughout eukaryotes. In plants, MAPKs are involved in biotic and abiotic stress responses; they regulate cell division, cell growth, and also programmed cell death. In vivo visualization of MAPKs is crucial for understanding of their spatiotemporal organization. Cloning of MAPK-fluorescent protein fusions might present difficulties related to the preservation of protein-protein interactions essential for MAPK localization, interactions with upstream and downstream regulators, and finally substrate targeting. In this chapter we describe cloning of MAPKs in the flexible MultiSite Gateway(®) cloning system followed by easy and quick testing of binary vectors by transient assays in Arabidopsis thaliana and Nicotiana benthamiana.

Evolutionary history of mitogen-activated protein kinase (MAPK) genes in Lotus, Medicago, and Phaseolus

Mitogen-Activated Protein Kinase (MAPK) genes encode proteins that mediate various signaling pathways associated with biotic and abiotic stress responses in eukaryotes. The MAPK genes form a 3-tier signal transduction cascade between cellular stimuli and physiological responses. Recent identification of soybean MAPKs and availability of genome sequences from other legume species allowed us to identify their MAPK genes. The main objectives of this study were to identify MAPKs in 3 legume species, Lotus japonicus, Medicago truncatula, and Phaseolus vulgaris, and to assess their phylogenetic relationships. We used approaches in comparative genomics for MAPK gene identification and named the newly identified genes following Arabidopsis MAPK nomenclature model. We identified 19, 18, and 15 MAPKs and 7, 4, and 9 MAPKKs in the genome of Lotus japonicus, Medicago truncatula, and Phaseolus vulgaris, respectively. Within clade placement of MAPKs and MAPKKs in the 3 legume species were consistent with those in soybean and Arabidopsis. Among 5 clades of MAPKs, 4 founder clades were consistent to MAPKs of other plant species and orthologs of MAPK genes in the fifth clade-"Clade E" were consistent with those in soybean. Our results also indicated that some gene duplication events might have occurred prior to eudicot-monocot divergence. Highly diversified MAPKs in soybean relative to those in 3 other legume species are attributable to the polyploidization events in soybean. The identification of the MAPK genes in the legume species is important for the legume crop improvement; and evolutionary relationships and functional divergence of these gene members provide insights into plant genome evolution.