MAP kináz jelátvitel funkcionális vizsgálat mitokondriumban = Functional studies on mitochondrial MAP kinase signalling

Munkánk során a növényekre specifikus, kevésbé vizsgált, D típusú MAPKok családjának egyik tagját az AtMPK9-t tanulmányoztuk. Élesztő kettős-hibrid rendszerrel a kalmodulint, mint lehetséges AtMPK9 fehérje partnert azonosítottuk, majd a kölcsönhatást in vitro transzlációval előállított fehérjékkel többféle megközelítéssel igazoltuk. Az AtMPK9 poszttranszlációs módosításokon keresztül történő szabályozása korábban ismeretlen volt. A pályázat keretében tömegspektrometriás vizsgálatokkal és in vitro mutagenezissel előállított AtMPK9 variánsokkal bizonyítottuk, hogy az aktiválásért felelős T hurok régióban elhelyezkedő TDY aminosav triplet treoninjának és tirozinjának foszforilálása nélkül a kináz nem rendelkezik aktivitással. A tömegspektrometriás adatok alapján az is nyilvánvaló vált, hogy az AtMPK9 kináz doménjét követő C-terminális doménben további négy aminosav foszforilálódik. Vizsgálataink szerint az összes általunk azonosított foszforiláció autofoszforiláció eredménye. Feltételezésünk szerint a kináz autofoszforilációs aktivitásának szabályozásában a kölcsönható partnerként azonosított kalmodulin kaphat szerepet. Az AtMPK9 in planta funkcióját protoplaszt tranziens expresszióval és null-mutáns növényekkel tanulmányoztuk. Vizsgálataink alapján a fehérje kináz abiotikus stresszel aktiválható, azonban ennek ellenére a null-mutáns növények fenotípusa még stressz körülmények között sem tér el a vadtípusétól, így az AtMPK9 valószínűsíthetően funkcionálisan redudáns kináz. | The project aimed at studying AtMPK9, a member of plant specific, D type mitogen activated protein kinase (MAPK). We identified calmodulin as its putative protein interacting partner by yeast two-hybrid assay. In order to evaluate this result, AtMPK9 and calmodulin were produced by in vitro translation and the interaction was confirmed by pull-down assays and surface plasmone resonance analysis. The kinase activity regulation of AtMPK9 was unknown previously. We demonstrated by mass spectrometry and in vitro mutagenesis studies that phosphorylation of threonine and tyrosine of TDY amino acid triad of T loop is inevitable for kinase activity. Further mass spectrometry analysis revealed another four phosphorylated amino acids in the C-terminal domain of AtMPK9. According to our in vitro translation based data, all the identified phosphorylations are caused by autophosphorylation. We hypothesize that the interacting partner calmodulin regulates the autophosphorylation activity of kinase. The in planta function of the protein kinase was studied by protoplast transient overexpression and application of AtMPK9 knock-out plants. Although the kinase activity of AtMPK9 was inducible by abiotic stress, the knock-out plants did not show any difference in phenotype, not even in stress conditions. These data imply that AtMPK9 is a functionally redundant protein kinase

The interplay between pathogens and Atg8 family proteins: thousand-faced interactions

Autophagy is an intracellular degradation and recycling process that can also remove pathogenic intracellular bacteria and viruses from within cells (referred to as xenophagy) and activate the adaptive immune responses. But autophagy - especially Atg proteins including Atg8 family members - can also have proviral and probacterial effects. In this review, we summarize known interactions of bacterial, parasitic and viral proteins with Atg8 family proteins and the outcome of these interactions on pathogen replication, autophagy or mitophagy. We discuss the value of prediction software and the research methodology in the study of pathogen protein-Atg8 family protein interactions, with selected examples of potential LIR motif-containing SARS-CoV-2 proteins

Satisfiability in multi-valued circuits

Satisfiability of Boolean circuits is among the most known and important problems in theoretical computer science. This problem is NP-complete in general but becomes polynomial time when restricted either to monotone gates or linear gates. We go outside Boolean realm and consider circuits built of any fixed set of gates on an arbitrary large finite domain. From the complexity point of view this is strictly connected with the problems of solving equations (or systems of equations) over finite algebras. The research reported in this work was motivated by a desire to know for which finite algebras $\mathbf A$ there is a polynomial time algorithm that decides if an equation over $\mathbf A$ has a solution. We are also looking for polynomial time algorithms that decide if two circuits over a finite algebra compute the same function. Although we have not managed to solve these problems in the most general setting we have obtained such a characterization for a very broad class of algebras from congruence modular varieties. This class includes most known and well-studied algebras such as groups, rings, modules (and their generalizations like quasigroups, loops, near-rings, nonassociative rings, Lie algebras), lattices (and their extensions like Boolean algebras, Heyting algebras or other algebras connected with multi-valued logics including MV-algebras). This paper seems to be the first systematic study of the computational complexity of satisfiability of non-Boolean circuits and solving equations over finite algebras. The characterization results provided by the paper is given in terms of nice structural properties of algebras for which the problems are solvable in polynomial time.Comment: 50 page

A rapid and robust assay for detection of S-phase cell cycle progression in plant cells and tissues by using ethynyl deoxyuridine

<p>Abstract</p> <p>Background</p> <p>Progress in plant cell cycle research is highly dependent on reliable methods for detection of cells replicating DNA. Frequency of S-phase cells (cells in DNA synthesis phase) is a basic parameter in studies on the control of cell division cycle and the developmental events of plant cells. Here we extend the microscopy and flow cytometry applications of the recently developed EdU (5-ethynyl-2'-deoxyuridine)-based S-phase assay to various plant species and tissues. We demonstrate that the presented protocols insure the improved preservation of cell and tissue structure and allow significant reduction in assay duration. In comparison with the frequently used detection of bromodeoxyuridine (BrdU) and tritiated-thymidine incorporation, this new methodology offers several advantages as we discuss here.</p> <p>Results</p> <p>Applications of EdU-based S-phase assay in microscopy and flow cytometry are presented by using cultured cells of alfalfa, Arabidopsis, grape, maize, rice and tobacco. We present the advantages of EdU assay as compared to BrdU-based replication assay and demonstrate that EdU assay -which does not require plant cell wall digestion or DNA denaturation steps, offers reduced assay duration and better preservation of cellular, nuclear and chromosomal morphologies. We have also shown that fast and efficient EdU assay can also be an efficient tool for dual parameter flow cytometry analysis and for quantitative assessment of replication in thick root samples of rice.</p> <p>Conclusions</p> <p>In plant cell cycle studies, EdU-based S-phase detection offers a superior alternative to the existing S-phase assays. EdU method is reliable, versatile, fast, simple and non-radioactive and it can be readily applied to many different plant systems.</p

A ferritin szerepe a szőlő stressztűrő képességének fokozásában = The role of ferritin in enhancing the stress tolerance of grapevine

Embriogén kalluszt állítottuk elő Richter110 (alany) és Chardonnay (nemes) szőlő fajták portokajiból. Ezeket Medicago sativa ferritint (MsFerr) tartalmazó Agrobacterium vektorokkal transzformáltunk. A DNS beépülését genomikus PCR-rel ellenőriztük. Poliklonális ellenanyagot állítottunk elő, mellyel megállapítottuk, hogy a transzláció magas szintű volt, a transzkriptumból nagy mennyiségű, megfelelően processzált fehérjetermék keletkezett. A transzgenikus szőlő növények elkészültéig dohányban teszteltük az MsFerr gént tartalmazó konstrukciókat. A teljes növényeken végzett UV-B kezelés, ill. levélkorongok közvetlen oxidatív stresszre adott válaszai alapján megállapítottuk, hogy az MsFerr növények toleránsabbak voltak, mint a nem expresszáló kontrollok. Az MsFerr Richter 110 növények regenerálása, szelekciója és felszaporítása sikeres volt, az MsFerr Chardonnay növények felszaporítása azonban hajtásnövekedési problémák miatt nem sikerült. Három MsFerr Richter 110 vonalat vizsgáltunk. Gyökér stresszként fás szárú dugványok tápoldatához hidrogénkarbonátot adtunk, ami klorotikus és levél száradási tünetek okozott Ebben nem kaptunk lényeges különbséget a transzgenikus és transzformálatlan növények között. Ezzel szemben, a leveleket érő hatásokkal: paraquat, NaCl só-stressz és tBHP indukált lipid peroxidációval szemben az MsFerr expresszáló vonalak toleránsabbak voltak (kisebb stressz-indukált fotoszintézis csökkenést mutattak) mint a transzformálatlanok. | Embryogenic calli were started from anthers of Richter 110 (rootstock) and Chardonnay (scion) grapevine and transformed with Agrobacterium harbouring Medicago sativa ferritin gene (MsFerr). Genomic PCR and protein immunoblotting using a polyclonal antibody confirmed that the transcription and processing of MsFerr was successful. Regeneration, selection and propagation of MsFerr Richter 110 plants was successful, but transformed MsFerr Chardonnay plants did not grow sufficiently. Until transgenic grapevine plants became available, preliminary experiments were carried out with MsFerr expressing tobacco. UV-B irradiation of whole plants as well as treatments of leaf disks with various chemical elicitors showed that MsFerr plants were more stress tolerant than non-expressing controls. After regeneration and propagation, three transgenic MsFerr Richter 110 lines were tested. Roots of green cuttings were stressed by flooding and in this experiment transgenic plants did not show significantly higher tolerance to hypoxia/bicarbonate than non-transferred ones. Leaves, however, showed increased tolerance to paraquat, salt stress and tBHP induced lipid peroxidation: their photosynthesis was less affected by these stressors than those from non-transformed plants

Chromosome numbers of selected species of Elatine L. (Elatinaceae)

The paper reports chromosome numbers for 13 taxa of Elatine L., including all 11 species occurring in Europe, namely E. alsinastrum, E. ambigua, E. brachysperma, E. brochonii, E. californica, E. campylosperma, E. gussonei, E. hexandra, E. hungarica, E. hydropiper, E. macropoda, E. orthosperma, E. triandra originating from 17, field-collected populations. For seven of them (E. ambigua, E. californica, E. campylosperma, E. brachysperma, E. brochonii, E. hungarica, E. orthosperma) the chromosome numbers are reported for the first time. With these records, chromosome numbers for the whole section Elatinella Seub. became available. Although 2n = 36 was reported to be the most common and the lowest chromosome number in the genus, our data show that out of thirteen species analyzed, six had 36 chromosomes but five species had 54 chromosomes, and the lowest number of chromosomes was 18. These data further corroborates that the basic chromosome number in Elatine is x = 9

Chromosome numbers of selected species of Elatine L. (Elatinaceae)

L

Activation of AtMPK9 through autophosphorylation that makes it independent of the canonical MAPK cascades.

Mitogen activated protein kinases (MAPKs) are part of conserved signal transduction modules in eukaryotes that are typically organised into three-tiered kinase cascades. The activation of MAPKs in these pathways is fully dependent on the bisphosphorylation of the TXY motif in the T-loop by the pertinent dual-specificity MAPK kinases (MAPKKs). The plant AtMPK9 is a member of an atypical class of MAPKs. Representatives of this MAPK family have TDY phosphoacceptor site, a long C-terminal extension, and lack the common MAPKK binding docking motif. Here, we present multiple in vitro and in vivo data that AtMPK9 is activated independently of any upstream MAPKKs but it is activated through autophosphorylation. We mapped the autophosphorylation sites by mass spectrometry to the TDY motif and to the C-terminal regulatory extension. We mutated the phosphoacceptor sites on the TDY, which confirmed the requirement for bisphorylation at this site for full kinase activity. Next, we demonstrated that the kinase inactive mutant form of AtMPK9 is not transphosphorylated on the TDY site when mixed with an active AtMPK9, implying that the mechanism of the autocatalytic phosphorylation is intramolecular. Furthermore, we show that in vivo AtMPK9 is activated by salt and is regulated by okadaic acid-sensitive phosphatases. We conclude that the plant AtMPK9 shows similarities to the mammalian atypical MAPKs, ERK7/8 in terms of MAPKK-independent activation mechanism

Imaging fluorescence detected linear dichroism of plant cell walls in laser scanning confocal microscope

Anisotropy carries important information on the molecular organization of biological samples. Its determination requires a combination of microscopy and polarization spectroscopy tools. The authors constructed differential polarization (DP) attachments; to a laser scanning microscope in order to determine physical quantities related to the anisotropic distribution of molecules in microscopic samples; here the authors focus on fluorescence-detected linear dichroism (FDLD). By modulating the linear polarization of the laser beam between two orthogonally polarized states and by using a demodulation circuit, the authors determine the associated transmitted and fluorescence intensity-difference signals, which serve the basis for LD (linear dichroism) and FDLD, respectively. The authors demonstrate on sections of Convallaria majalis root tissue stained with Acridin Orange that while (nonconfocal) LD images remain smeared and weak, FDLD images recorded in confocal mode reveal strong anisotropy of the cell wall. FDLD imaging is suitable for mapping the anisotropic distribution of transition dipoles in 3 dimensions. A mathematical model is proposed to account for the fiber-laminate ultrastructure of the cell wall and for the intercalation of the dye molecules in complex, highly anisotropic architecture. (c) 2007 International Society for Analytical Cytology