Arabidopsis MPK4 and MPK6 are implicated in different signalling pathways responding to diverse external stimuli. This was recently correlated with transcriptomic profiles of Arabidopsis mpk4 and mpk6 mutants, and thus it should be reflected also on the level of constitutive proteomes. Therefore, we performed a shot gun comparative proteomic analysis of Arabidopsis mpk4 and mpk6 mutant roots. We have used bioinformatic tools and propose several new proteins as putative MPK4 and MPK6 phosphorylation targets. Among these proteins in the mpk6 mutant were important modulators of development such as CDC48A and phospholipase D alpha 1. In the case of the mpk4 mutant transcriptional reprogramming might be mediated by phosphorylation and change in the abundance of mRNA decapping complex VCS. Further comparison of mpk4 and mpk6 root differential proteomes showed differences in the composition and regulation of defense related proteins. The mpk4 mutant showed altered abundances of antioxidant proteins. The examination of catalase activity in response to oxidative stress revealed that this enzyme might be preferentially regulated by MPK4. Finally, we proposed developmentally important proteins as either directly or indirectly regulated by MPK4 and MPK6. These proteins contribute to known phenotypic defects in the mpk4 and mpk6 mutants.

• MeSH
• Arabidopsis enzymologie   genetika   MeSH
• fosforylace MeSH
• fyziologický stres MeSH
• genová ontologie MeSH
• genový knockout MeSH
• katalasa metabolismus   MeSH
• kořeny rostlin enzymologie   genetika   MeSH
• missense mutace MeSH
• mitogenem aktivované proteinkinasy genetika   MeSH
• peroxidasa metabolismus   MeSH
• posttranslační úpravy proteinů MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• proteom metabolismus   MeSH
• proteomika MeSH
• receptory pro aktivovanou kinasu C metabolismus   MeSH
• sekvence aminokyselin MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• srovnávací studie MeSH

The role of YODA MITOGEN ACTIVATED PROTEIN KINASE KINASE KINASE 4 (MAPKKK4) upstream of MITOGEN ACTIVATED PROTEIN KINASE 6 (MPK6) was studied during post-embryonic root development of Arabidopsis thaliana. Loss- and gain-of-function mutants of YODA (yda1 and ΔNyda1) were characterized in terms of root patterning, endogenous auxin content and global proteomes. We surveyed morphological and cellular phenotypes of yda1 and ΔNyda1 mutants suggesting possible involvement of auxin. Endogenous indole-3-acetic acid (IAA) levels were up-regulated in both mutants. Proteomic analysis revealed up-regulation of auxin biosynthetic enzymes tryptophan synthase and nitrilases in these mutants. The expression, abundance and phosphorylation of MPK3, MPK6 and MICROTUBULE ASSOCIATED PROTEIN 65-1 (MAP65-1) were characterized by quantitative polymerase chain reaction (PCR) and western blot analyses and interactions between MAP65-1, microtubules and MPK6 were resolved by quantitative co-localization studies and co-immunoprecipitations. yda1 and ΔNyda1 mutants showed disoriented cell divisions in primary and lateral roots, abortive cytokinesis, and differential subcellular localization of MPK6 and MAP65-1. They also showed deregulated expression of TANGLED1 (TAN1), PHRAGMOPLAST ORIENTING KINESIN 1 (POK1), and GAMMA TUBULIN COMPLEX PROTEIN 4 (GCP4). The findings that MPK6 localized to preprophase bands (PPBs) and phragmoplasts while the mpk6-4 mutant transformed with MPK6AEF (alanine (A)-glutamic acid (E)-phenylanine (F)) showed a root phenotype similar to that of yda1 demonstrated that MPK6 is an important player downstream of YODA. These data indicate that YODA and MPK6 are involved in post-embryonic root development through an auxin-dependent mechanism regulating cell division and mitotic microtubule (PPB and phragmoplast) organization.

• MeSH
• Arabidopsis cytologie   účinky léků   embryologie   enzymologie   MeSH
• buněčné dělení * účinky léků   MeSH
• cytokineze účinky léků   MeSH
• epidermis rostlin cytologie   MeSH
• fenotyp MeSH
• fluorescenční protilátková technika MeSH
• fosforylace účinky léků   MeSH
• interfáze MeSH
• kořeny rostlin anatomie a histologie   cytologie   embryologie   MeSH
• kyseliny indoloctové metabolismus   farmakologie   MeSH
• MAP kinasy kinas (kinas) metabolismus   MeSH
• meristém cytologie   účinky léků   MeSH
• mikrotubuly účinky léků   metabolismus   MeSH
• mitogenem aktivované proteinkinasy metabolismus   MeSH
• mitóza účinky léků   MeSH
• mutace genetika   MeSH
• proteiny huseníčku metabolismus   MeSH
• proteomika MeSH
• transport proteinů účinky léků   MeSH
• upregulace * účinky léků   MeSH
• vazba proteinů účinky léků   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Stress-activated plant mitogen-activated protein (MAP) kinase pathways play roles in growth adaptation to the environment by modulating cell division through cytoskeletal regulation, but the mechanisms are poorly understood. We performed protein interaction and phosphorylation experiments with cytoskeletal proteins, mass spectrometric identification of MPK6 complexes and immunofluorescence analyses of the microtubular cytoskeleton of mitotic cells using wild-type, mpk6-2 mutant and plants overexpressing the MAP kinase-inactivating phosphatase, AP2C3. We showed that MPK6 interacted with γ-tubulin and co-sedimented with plant microtubules polymerized in vitro. It was the active form of MAP kinase that was enriched with microtubules and followed similar dynamics to γ-tubulin, moving from poles to midzone during the anaphase-to-telophase transition. We found a novel substrate for MPK6, the microtubule plus end protein, EB1c. The mpk6-2 mutant was sensitive to 3-nitro-l-tyrosine (NO2 -Tyr) treatment with respect to mitotic abnormalities, and root cells overexpressing AP2C3 showed defects in chromosome segregation and spindle orientation. Our data suggest that the active form of MAP kinase interacts with γ-tubulin on specific subsets of mitotic microtubules during late mitosis. MPK6 phosphorylates EB1c, but not EB1a, and has a role in maintaining regular planes of cell division under stress conditions.

• MeSH
• anafáze účinky léků   MeSH
• aparát dělícího vřeténka účinky léků   metabolismus   MeSH
• Arabidopsis cytologie   účinky léků   enzymologie   MeSH
• butadieny farmakologie   MeSH
• cytokineze účinky léků   MeSH
• extracelulárním signálem regulované MAP kinasy metabolismus   MeSH
• fosforylace účinky léků   MeSH
• fyziologický stres * účinky léků   MeSH
• kinetochory účinky léků   metabolismus   MeSH
• meristém cytologie   účinky léků   metabolismus   MeSH
• mikrotubuly účinky léků   metabolismus   MeSH
• mitogenem aktivované proteinkinasy metabolismus   MeSH
• nitrily farmakologie   MeSH
• nitrosace účinky léků   MeSH
• proliferace buněk účinky léků   MeSH
• proteiny asociované s mikrotubuly metabolismus   MeSH
• proteiny huseníčku metabolismus   MeSH
• rostlinné buňky účinky léků   metabolismus   MeSH
• segregace chromozomů účinky léků   MeSH
• telofáze účinky léků   MeSH
• tubulin metabolismus   MeSH
• tyrosin analogy a deriváty   farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Stomatal ontogenesis, patterning, and function are hallmarks of environmental plant adaptation, especially to conditions limiting plant growth, such as elevated temperatures and reduced water availability. The specification and distribution of a stomatal cell lineage and its terminal differentiation into guard cells require a master regulatory protein phosphorylation cascade involving the YODA mitogen-activated protein kinase kinase kinase. YODA signaling results in the activation of MITOGEN-ACTIVATED PROTEIN KINASEs (MPK3 and MPK6), which regulate transcription factors, including SPEECHLESS (SPCH). Here, we report that acute heat stress affects the phosphorylation and deactivation of SPCH and modulates stomatal density. By using complementary molecular, genetic, biochemical, and cell biology approaches, we provide solid evidence that HEAT SHOCK PROTEINS 90 (HSP90s) play a crucial role in transducing heat-stress response through the YODA cascade. Genetic studies revealed that YODA and HSP90.1 are epistatic, and they likely function linearly in the same developmental pathway regulating stomata formation. HSP90s interact with YODA, affect its cellular polarization, and modulate the phosphorylation of downstream targets, such as MPK6 and SPCH, under both normal and heat-stress conditions. Thus, HSP90-mediated specification and differentiation of the stomatal cell lineage couples stomatal development to environmental cues, providing an adaptive heat stress response mechanism in plants.

• MeSH
• Arabidopsis fyziologie   MeSH
• buněčná diferenciace MeSH
• buněčné dělení MeSH
• buněčný rodokmen MeSH
• epigeneze genetická MeSH
• fosforylace MeSH
• kotyledon cytologie   MeSH
• MAP kinasy kinas (kinas) genetika   metabolismus   MeSH
• mitogenem aktivované proteinkinasy kinas metabolismus   MeSH
• mitogenem aktivované proteinkinasy metabolismus   MeSH
• mutace MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• proteiny tepelného šoku HSP90 genetika   metabolismus   MeSH
• průduchy rostlin cytologie   růst a vývoj   metabolismus   MeSH
• reakce na tepelný šok * MeSH
• regulace genové exprese u rostlin MeSH
• signální transdukce MeSH
• transkripční faktory bHLH metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Dual-specificity mitogen-activated protein kinases kinases (MAPKKs) are the immediate upstream activators of MAPKs. They simultaneously phosphorylate the TXY motif within the activation loop of MAPKs, allowing them to interact with and regulate multiple substrates. Often, the activation of MAPKs triggers their nuclear translocation. However, the spatiotemporal dynamics and the physiological consequences of the activation of MAPKs, particularly in plants, are still poorly understood. Here, we studied the activation and localization of the Medicago sativa stress-induced MAPKK (SIMKK)-SIMK module after salt stress. In the inactive state, SIMKK and SIMK co-localized in the cytoplasm and in the nucleus. Upon salt stress, however, a substantial part of the nuclear pool of both SIMKK and SIMK relocated to cytoplasmic compartments. The course of nucleocytoplasmic shuttling of SIMK correlated temporally with the dual phosphorylation of the pTEpY motif. SIMKK function was further studied in Arabidopsis plants overexpressing SIMKK-yellow fluorescent protein (YFP) fusions. SIMKK-YFP plants showed enhanced activation of Arabidopsis MPK3 and MPK6 kinases upon salt treatment and exhibited high sensitivity against salt stress at the seedling stage, although they were salt insensitive during seed germination. Proteomic analysis of SIMKK-YFP overexpressors indicated the differential regulation of proteins directly or indirectly involved in salt stress responses. These proteins included catalase, peroxiredoxin, glutathione S-transferase, nucleoside diphosphate kinase 1, endoplasmic reticulum luminal-binding protein 2, and finally plasma membrane aquaporins. In conclusion, Arabidopsis seedlings overexpressing SIMKK-YFP exhibited higher salt sensitivity consistent with their proteome composition and with the presumptive MPK3/MPK6 hijacking of the salt response pathway.

• MeSH
• aktivace enzymů MeSH
• Arabidopsis genetika   růst a vývoj   metabolismus   MeSH
• exprese genu MeSH
• geneticky modifikované rostliny genetika   růst a vývoj   metabolismus   MeSH
• Medicago sativa enzymologie   genetika   MeSH
• mitogenem aktivované proteinkinasy kinas genetika   metabolismus   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• semenáček genetika   růst a vývoj   metabolismus   MeSH
• soli metabolismus   MeSH
• transport proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

In all eukaryotes, signaling by mitogen-activated protein kinase (MAPK) pathways plays a crucial role in signal transduction during regulation of cell growth, differentiation, proliferation as well as death and stress responses. In this chapter we describe a reliable method to immunolocalize MAPKs in roots of Arabidopsis thaliana by using whole-mount seedling probes. This method relies on quick and efficient chemical fixation, partial cell wall digestion, plasma membrane permeabilization, subsequent antibody incubation, and visualization by high-end confocal laser scanning microscopy (CLSM) performed on whole Arabidopsis seedlings. Protocols are provided for immunofluorescent localization of MPK3, MPK4, and MPK6, representing three major developmentally and stress-regulated MAPKs of Arabidopsis. In addition, protocols for colocalization of these MAPKs with microtubules are also provided.

• MeSH
• Arabidopsis cytologie   enzymologie   růst a vývoj   metabolismus   MeSH
• fluorescenční protilátková technika metody   MeSH
• konfokální mikroskopie MeSH
• mikrotubuly metabolismus   MeSH
• mitogenem aktivované proteinkinasy analýza   metabolismus   MeSH
• semenáček cytologie   enzymologie   růst a vývoj   MeSH
• transport proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH