Phospholipase Dα1 (PLDα1) belongs to phospholipases, a large phospholipid hydrolyzing protein family. PLDα1 has a substrate preference for phosphatidylcholine leading to enzymatic production of phosphatidic acid, a lipid second messenger with multiple cellular functions. PLDα1 itself is implicated in biotic and abiotic stress responses. Here, we present a shot-gun differential proteomic analysis on roots of two Arabidopsis pldα1 mutants compared to the wild type. Interestingly, PLDα1 deficiency leads to altered abundances of proteins involved in diverse processes related to membrane transport including endocytosis and endoplasmic reticulum-Golgi transport. PLDα1 may be involved in the stability of attachment sites of endoplasmic reticulum to the plasma membrane as suggested by increased abundance of synaptotagmin 1, which was validated by immunoblotting and whole-mount immunolabelling analyses. Moreover, we noticed a robust abundance alterations of proteins involved in mitochondrial import and electron transport chain. Notably, the abundances of numerous proteins implicated in glucosinolate biosynthesis were also affected in pldα1 mutants. Our results suggest a broader biological involvement of PLDα1 than anticipated thus far, especially in the processes such as endomembrane transport, mitochondrial protein import and protein quality control, as well as glucosinolate biosynthesis.

• MeSH
• Arabidopsis metabolismus   MeSH
• endocytóza MeSH
• fosfolipasa D genetika   metabolismus   MeSH
• genová ontologie MeSH
• glukosinoláty biosyntéza   MeSH
• kořeny rostlin metabolismus   MeSH
• mitochondriální proteiny metabolismus   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• proteom metabolismus   MeSH
• proteomika * MeSH
• synaptotagmin I metabolismus   MeSH
• tandemová hmotnostní spektrometrie MeSH
• transport proteinů MeSH
• uncoupling protein 1 metabolismus   MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Publikační typ
• časopisecké články MeSH

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

Disentanglement of functional complexity associated with plant mitogen-activated protein kinase (MAPK) signaling has benefited from transcriptomic, proteomic, phosphoproteomic, and genetic studies. Published transcriptomic analysis of a double homozygous recessive anp2anp3 mutant of two MAPK kinase kinase (MAPKKK) genes called Arabidopsis thaliana Homologues of Nucleus- and Phragmoplast-localized Kinase 2 (ANP2) and 3 (ANP3) showed the upregulation of stress-related genes. In this study, a comparative proteomic analysis of anp2anp3 mutant against its respective Wassilevskaja ecotype (Ws) wild type background is provided. Such differential proteomic analysis revealed overabundance of core enzymes such as FeSOD1, MnSOD, DHAR1, and FeSOD1-associated regulatory protein CPN20, which are involved in the detoxification of reactive oxygen species in the anp2anp3 mutant. The proteomic results were validated at the level of single protein abundance by Western blot analyses and by quantitative biochemical determination of antioxidant enzymatic activities. Finally, the functional network of proteins involved in antioxidant defense in the anp2anp3 mutant was physiologically linked with the increased resistance of mutant seedlings against paraquat treatment.

• MeSH
• antioxidancia metabolismus   MeSH
• Arabidopsis účinky léků   genetika   metabolismus   MeSH
• biologické modely MeSH
• chromatografie kapalinová MeSH
• herbicidy farmakologie   MeSH
• imunoblotting MeSH
• MAP kinasy kinas (kinas) genetika   metabolismus   MeSH
• mutace MeSH
• paraquat farmakologie   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• proteom genetika   metabolismus   MeSH
• proteomika metody   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• semenáček genetika   metabolismus   účinky léků   MeSH
• signální transdukce účinky léků   MeSH
• superoxiddismutasa metabolismus   MeSH
• tandemová hmotnostní spektrometrie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. 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

Protein phosphorylation is the most abundant and best studied protein posttranslational modification, dedicated to the regulation of protein function and subcellular localization as well as to protein-protein interactions. Identification and quantitation of the dynamic, conditional protein phosphorylation can be achieved by either metabolic labeling of the protein of interest with (32)P-labeled ATP followed by autoradiographic analysis, the use of specific monoclonal or polyclonal antibodies against the phosphorylated protein species and finally by phosphoproteome delineation using mass spectrometry.Hereby we present a fourth alternative which relies on the enforced-affinity-based-electrophoretic separation of phosphorylated from non-phosphorylated protein species by standard SDS-PAGE systems co-polymerized with Phos-Tag™ and Mn(2+) or Zn(2+) cations. Phosphate groups of phosphorylated Ser, Thr, and Tyr residues form complexes with Mn(2+) and Zn(2+) cations with polyacrylamide immobilized Phos-Tag™. Following appropriate treatment of the gels, separated proteins can be quantitatively transferred to PVDF or nitrocellulose membranes and probed with common-not phosphorylation state specific-antibodies and delineate the occurrence of a certain phosphoprotein species against its non-phosphorylated counterpart.

• MeSH
• akrylamid chemie   MeSH
• Arabidopsis enzymologie   růst a vývoj   MeSH
• bakteriofág lambda enzymologie   MeSH
• elektroforéza v polyakrylamidovém gelu metody   MeSH
• fenol chemie   MeSH
• fosfatasy metabolismus   MeSH
• fosfoproteiny izolace a purifikace   metabolismus   MeSH
• fosforylace MeSH
• kultivační techniky MeSH
• membrány umělé MeSH
• mitogenem aktivované proteinkinasy izolace a purifikace   metabolismus   MeSH
• polyvinyly chemie   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

• Publikační typ
• abstrakt z konference MeSH