3D structure prediction of histone acetyltransferase proteins of the MYST family and their interactome in Arabidopsis thaliana.

Histone lysine acetylation is a reversible post-translational modification that does not involve changes in DNA sequences. Enzymes play an important role in developmental processes and their deregulation has been linked to the progression of diverse disorders. The HAT enzyme family fulfills an important role in various developmental processes mediated by the state of chromatin, and have been attributed to its deregulation. To understand acetylation mechanisms and their role in cell signaling, transcriptional regulation, and apoptosis, it is crucial to identify and analyze acetylation sites. Bioinformatics methods can be used to generate relatively precise predictions. Here we applied classical bioinformatics methods-sequence alignment, homology modeling, and docking-to compare approved and predicted lysine acetylation processes in different organisms. HAM1 and HAM2 are analogs of KAT8 and KAT7 (MYST1 and MYST2), members of the MYST histone acetyltransferase family, and our results show that HAM1 and HAM2 have much in common with other representatives of MYST families from various organisms. One function of acetyl-CoA binding was predicted with a high level of probability by computational methods. Based on our data, we conclude that, despite huge genetic distances and some structural differences between animal and plant species, a closer look at acetylation mechanism shows that they have much in common.

[Efficiency of the Induction of Cytomixis in the Microsporogenesis of Dicotyledonous (N. tabacum L.) and Monocotyledonous (H. distichum L.) Plants by Thermal Stress].

The efficiencies of the induction of cytomixis in microsporogenesis by thermal stress are compared in tobacco (N. tabacum L.) and barley (H. distichum L.) It has been shown that different thermal treatment schedules (budding tobacco plants at 50°C and air-dried barley grains at 48°C) produce similar results in the species: the frequency of cytomixis increases, and its maximum shifts to later stages of meiosis. However, the species show differences in response. The cytomixis frequency increase in tobacco is more pronounced, and its maximum shifts from the zygotene­pachytene stages of meiotic prophase I to prometaphase­metaphase I. Later in the meiosis, aberrations in chromosome structure and meiotic apparatus formation typical of cytomixis are noted, as well as cytomixis activation in tapetum cells. Thermal stress disturbs the integration of callose- bearing vesicles into the callose wall. Cold treatment at 7°C does not affect cytomixis frequency in tobacco microsporogenesis. Incubation of barley seeds at 48°C activates cytomixis in comparison to the control, shifts its maximum from the premeiotic interphase to zygotene, and changes the habit of cytomictic interactions from pairwise contacts to the formation of multicellular clusters. Thermal treatment induces cytomictic interactions within the tapetum and between microsporocytes and the tapetum. However, later meiotic phases show no adverse consequences of active cytomixis in barley. It is conjectured that heat stress affects callose metabolism and integration into the forming callose wall, thereby causing incomplete closure of cytomictic channels and favoring intercellular chromosome migration at advanced meiotic stages.

Efficient callus formation and plant regeneration of goosegrass [Eleusine indica (L.) Gaertn.].

Efficient methods in totipotent callus formation, cell suspension culture establishment and whole-plant regeneration have been developed for the goosegrass [ Eleusine indica (L.) Gaertn.] and its dinitroaniline-resistant biotypes. The optimum medium for inducing morphogenic calli consisted of N6 basal salts and B5 vitamins supplemented with 1-2 mg l(-1) 2,4-dichlorophenoxyacetic acid (2,4-D), 2 mg l(-1) glycine, 100 mg l(-1) asparagine, 100 mg l(-1) casein hydrolysate, 30 g l(-1) sucrose and 0.6% agar, pH 5.7. The presence of organogenic and embryogenic structures in these calli was histologically documented. Cell suspension cultures derived from young calli were established in a liquid medium with the same composition. Morphogenic structures of direct shoots and somatic embryos were grown into rooted plantlets on medium containing MS basal salts, B5 vitamins, 1 mg l(-1) kinetin (Kn) and 0.1 mg l(-1) indole-3-acetic acid (IAA), 3% sucrose, 0.6% agar, pH 5.7. Calli derived from the R-biotype of E. indica possessed a high resistance to trifluralin (dinitroaniline herbicide) and cross-resistance to a structurally non-related herbicide, amiprophosmethyl (phosphorothioamidate herbicide), as did the original resistant plants. Embryogenic cell suspension culture was a better source of E. indica protoplasts than callus or mesophyll tissue. The enzyme solution containing 1.5% cellulase Onozuka R-10, 0.5% driselase, 1% pectolyase Y-23, 0.5% hemicellulase and N(6) mineral salts with an additional 0.2 M KCl and 0.1 M CaCl(2) (pH 5.4-5.5) was used for protoplast isolation. The purified protoplasts were cultivated in KM8p liquid medium supplemented with 2 mg l(-1) 2,4-D and 0.2 mg l(-1) Kn.

Exposure of tubulin structural domains in Nicotiana tabacum microtubules probed by monoclonal antibodies.

A panel of nine antibodies, specific to antigenic determinants located on N- or C-terminal structural domains of alpha and beta subunits of animal tubulin, and antibodies against acetylated, tyrosinated and polyglutamylated tubulins were utilized for probing the Nicotiana tabacum microtubules. The specificity of antibodies was confirmed by immunoblotting on whole cell lysates and on tubulin isoforms separated by high-resolution isoelectric focusing. Whereas antibodies TU-01 and TU-09 reacted with all alpha-tubulin isoforms and TU-06 reacted with all beta-tubulin isoforms, the other antibodies reacted with a limited number of tubulin isoforms. Antibody TU-14 reacted only with two beta-tubulin charge variants. In fixed cells, each of the antibodies stained microtubules of preprophase band, mitotic spindle and phragmoplast. Cortical microtubules were stained by all antibodies except TU-02 and TU-03, which did not decorate microtubules in interphase cells. Immunostaining of unfixed detergent-extracted cells revealed that antibodies against determinants on the C-terminal domains of both subunits decorated microtubules, but these were not stained with antibodies to determinants on the N-terminal domains. These data indicate that in plant microtubules at least several parts of the N-terminal domains of both subunits are either not exposed on the microtubule surface or are masked by the other proteins. In contrast, parts of the C-terminal domains are exposed on the exterior of microtubules. As for animal tubulins the majority of posttranslational modifications as well as binding sites for microtubule-associated proteins (MAPs) have been located to these regions, it is possible also in higher plants that the C-terminal structural domains of both tubulin subunits participate in the modulation of tubulin interactions with associated proteins.

Post-translational modifications and multiple tubulin isoforms in Nicotiana tabacum L. cells.

Distribution of post-translationally modified tubulins in cells of Nicotiana tabacum L. was analysed using a panel of specific antibodies. Polyglutamylated, tyrosinated, nontyrosinated, acetylated and delta 2-tubulin variants were detected on alpha-tubulin subunits; polyglutamylation was also found on beta-tubulin subunits. Modified tubulins were detected by immunofluorescence microscopy in interphase microtubules, preprophase bands, mitotic spindles as well as in phragmoplasts. They were, however, located differently in the various microtubule structures. The antibodies against tyrosinated, acetylated and polyglutamylated tubulins gave uniform staining along all microtubules, while antibodies against nontyrosinated and delta 2-tubulin provided dot-like staining of interphase microtubules. Additionally, immunoreactivity of antibodies against acetylated and delta 2-tubulins was strong in the pole regions of mitotic spindles. High-resolution isoelectric focusing revealed 22 tubulin charge variants in N. tabacum suspension cells. Immunoblotting with antibodies TU-01 and TU-06 against conserved antigenic determinants of alpha- and beta-tubulin molecules, respectively, revealed that 11 isoforms belonged to the alpha-subunit and 11 isoforms to the beta-subunit. Whereas antibodies against polyglutamylated, tyrosinated and acetylated tubulins reacted with several alpha-tubulin isoforms, antibodies against nontyrosinated and delta 2-tubulin reacted with only one. The combined data demonstrate that plant tubulin is extensively post-translationally modified and that these modifications participate in the generation of plant tubulin polymorphism.