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Background Cell-free protein expression has become a widely used alternative of in vivo, cell-based systems in functional and structural studies of proteins. The wheat germ-based method outstands from the commercially available eukaryotic in vitro translation systems by its flexibility, high translation efficiency and success rate of properly folded eukaryotic protein synthesis. The original T7 promoter containing pEU3-NII vector was improved previously by addition of a ligation-independent cloning site, His6- and GST-tags, and a TEV protease cleavage site to facilitate the creation of recombinant plasmids, permit affinity purification, and enable production of purified, tag-free target proteins, respectively. Results Here, we describe a further development of pEU3-NII vector by inserting the rare-cutting, NotI restriction enzyme cleavage site to simplify vector linearization step prior to in vitro transcription. Additionally, His12, FLAG, and Halo affinity tag coding vectors have been created to increase detection sensitivity, specificity of interaction studies, and provide covalently linkable ligands for pull-down assays, respectively. Finally, the presented GST-His6, and GST-biotin double-tagging vectors could broaden the range of possibilities of protein-protein interaction studies. Conclusions The new generation of pEU3-NII vector family allows a more rapid production of translationally active mRNA and wheat germ cell-free expression of target proteins with a wide variety of affinity tags thus enables designing flexible and diverse experimental arrangement for in vitro studies of proteins.

Plant growth flexibly adapts to environmental conditions, implying cross‐talk between environmental signalling and developmental regulation. Here, we show that the PIN auxin efflux carrier family possesses three highly conserved putative mitogen‐activated protein kinase (MAPK) sites adjacent to the phosphorylation sites of the well‐characterised AGC kinase PINOID, which regulates the polar localisation of PINs and directional auxin transport, thereby underpinning organ growth. The conserved sites of PIN1 are phosphorylated in vitro by two environmentally activated MAPKs, MPK4 and MPK6. In contrast to AGC kinases, MAPK‐mediated phosphorylation of PIN1 at adjacent sites leads to a partial loss of the plasma membrane localisation of PIN1. MAPK‐mediated modulation of PIN trafficking may participate in environmental adjustment of plant growth.

Oxidation stress induced by nanoparticles is considered to be the main mechanism of their toxicity. An evaluation of oxidative damage to DNA, one of the mostly endangered molecules, belongs to general approaches used in nanomaterials cytotoxicology. However, for spherical, biochemical and other reasons, they should be differences in nanoparticles behaviour using tests based on cells and pure nucleic acid substances. The goal of the work is to detect structural damage to isolated salmon sperm double stranded DNA at its exposure to UV-C radiation (254 nm) in the presence of thiol-capped CdS quantum dots of various size and fluorescence intensity. An electrochemical DNA-based biosensor composed of a glassy carbon electrode with a layered structure of chemical modifiers dsDNA and CdS QDs was prepared and used for the voltammetric and impedimetric evaluation of a DNA portion survived after the irradiation. The cyclic voltammetric response of the redox indicator system hexacyanoferrate(III/II) and square wave voltammetric response of the guanine and adenine moieties were measured as analytical signals. The results were verified by experiments with irradiation of DNA in the solution phase followed by voltammetric measurement of immobilized survived portion of DNA (biosensing) and by conventional agarose gel electrophoresis. Experiments showed significant effect of the fluorescent active quantum dots on the degree of DNA degradation.

γ-Tubulin is associated with microtubule nucleation, but evidence is accumulating in eukaryotes that it functions also in nuclear processes and in cell division control that are independent of its canonical role. We found that in Arabidopsis thaliana γ-tubulin interacts specifically with E2FA, E2FB, and E2FC transcription factors both in vitro and in vivo. The interaction of γ-tubulin with the E2Fs is not reduced in the presence of the dimerization partners (DPs) and, in agreement, we found that γ-tubulin interaction with E2Fs does not require the dimerization domain. γ-Tubulin associates with the promoters of E2F-regulated cell cycle genes in an E2F dependent manner, likely in complex with the E2F/DP heterodimer. The upregulation of E2F targets; PCNA, ORC2, CDKB1;1 and CCS52A under γtubulin silencing suggests a repressive function for γ-tubulin at G1/S, G2/M and endocycle, which is consistent with an excess of cell division in some cells and enhanced endoreduplication in others in the shoot and young leaves of γ-tubulin RNAi plants. Altogether, our data show ternary interaction of γ-tubulin with E2F/DP heterodimer and suggest a repressive role for γ-tubulin with E2Fs in controlling mitotic activity and endoreduplication during plant development.

γ-Tubulin is associated with microtubule nucleation, but evidence is accumulating in eukaryotes that it also functions in nuclear processes and in cell division control independently of its canonical role. We found that in Arabidopsis thaliana, γ-tubulin interacts specifically with E2FA, E2FB, and E2FC transcription factors both in vitro and in vivo. The interaction of γ-tubulin with the E2Fs is not reduced in the presence of their dimerization partners (DPs) and, in agreement, we found that γ-tubulin interaction with E2Fs does not require the dimerization domain. γ-Tubulin associates with the promoters of E2F-regulated cell cycle genes in an E2F-dependent manner, probably in complex with the E2F-DP heterodimer. The up-regulation of E2F target genes PCNA, ORC2, CDKB1;1, and CCS52A under γ-tubulin silencing suggests a repressive function for γ-tubulin at G1/S and G2/M transitions, and the endocycle, which is consistent with an excess of cell division in some cells and enhanced endoreduplication in others in the shoot and young leaves of γ-tubulin RNAi plants. Altogether, our data show ternary interaction of γ-tubulin with the E2F-DP heterodimer and suggest a repressive role for γ-tubulin with E2Fs in controlling mitotic activity and endoreduplication during plant development.

Mitogen-activated protein kinases (MAPKs) are part of conserved signal transduction modules in eukaryotes that are typically organized 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 Arabidopsis mitogen-activated protein kinase 9 (AtMPK9) is a member of an atypical class of MAPKs. Representatives of this MAPK family have a TDY phosphoacceptor site, a long C-terminal extension and lack the common MAPKK-binding docking motif. In the present paper, we describe multiple in vitro and in vivo data showing that AtMPK9 is activated independently of any upstream MAPKKs but rather is activated through autophosphorylation. We mapped the autophosphorylation sites by MS 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 trans-phosphorylated 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, such as extracellular-signal-regulated kinase (ERK) 7/8, in terms of an MAPKK-independent activation mechanism.