Exposure of beta-tubulin regions defined by antibodies on an Arabidopsis thaliana microtubule protofilament model and in the cells

<p>Abstract</p> <p>Background</p> <p>The function of the cortical microtubules, composed of αβ-tubulin heterodimers, is linked to their organizational state which is subject to spatial and temporal modulation by environmental cues. The role of tubulin posttranslational modifications in these processes is largely unknown. Although antibodies against small tubulin regions represent useful tool for studying molecular configuration of microtubules, data on the exposure of tubulin epitopes on plant microtubules are still limited.</p> <p>Results</p> <p>Using homology modeling we have generated an <it>Arabidopsis thaliana </it>microtubule protofilament model that served for the prediction of surface exposure of five β-tubulin epitopes as well as tyrosine residues. Peptide scans newly disclosed the position of epitopes detected by antibodies 18D6 (β1-10), TUB2.1 (β426-435) and TU-14 (β436-445). Experimental verification of the results by immunofluorescence microscopy revealed that the exposure of epitopes depended on the mode of fixation. Moreover, homology modeling showed that only tyrosines in the C-terminal region of β-tubulins (behind β425) were exposed on the microtubule external side. Immunofluorescence microscopy revealed tyrosine phosphorylation of microtubules in plant cells, implying that β-tubulins could be one of the targets for tyrosine kinases.</p> <p>Conclusions</p> <p>We predicted surface exposure of five β-tubulin epitopes, as well as tyrosine residues, on the surface of <it>A. thaliana </it>microtubule protofilament model, and validated the obtained results by immunofluorescence microscopy on cortical microtubules in cells.</p> <p>The results suggest that prediction of epitope exposure on microtubules by means of homology modeling combined with site-directed antibodies can contribute to a better understanding of the interactions of plant microtubules with associated proteins.</p

Increase in the Synthesis of Polyfructan in the Cultures of Chicory “Hairy Roots” with Plant Natural Growth Regulators

Experiments were conducted to study the benefit of using the new plant growth regulators (PGRs) Ivin, Emistim, Biolan and Charkor in nutrient medium ½ MS for intensification of biomass growth and the increased synthesis of polyfructan (PF) in the cultures of chicory “hairy roots” (Cichorium intybus L.), obtained by Аgrobacterium rhizogenes-mediated transformation,. The best indexes of increased specific quantities of PF are observed after using Biolan at a concentration of 5.0 μL/L (up to 130 mg/g dry mass of roots) and Emistim at a concentration 2.5 µL/L (up to 220 mg/g dry mass of roots). The greatest stimulation of root growth activity was expressed on using the growth regulators Emistim, Ivin and Charkor, in concentrations between 2.5 and 10.0 µL/L, considerably raising the total quantity of PF: compared with the control the use of regulator Emistim showed a rise of up to 35 times, regulator Ivin showed an increase of up to 28 times and regulator Charkor showed an increase up to 7.0-7.5 times. The results thus obtained definitely prove the benefit of applying these regulators to increase the biomass growth and PF synthesis in the culture of chicory «hairy roots»

Tubulin acetylation accompanies autophagy development induced by different abiotic stimuli in <em>arabidopsis thaliana</em>

Microtubules (MTs) play an important role in the regulation of autophagy development in yeast and animal as well as in plant cells. MTs participate in maturation and traffic of autophagosomes through their dynamic state changes and post-translational modifications of tubulin, namely acetylation. We subjected Arabidopsis thaliana seedlings to metabolic-, salt-, osmotic stresses as well as irradiation of ultraviolet B and investigated the involvement of plant MTs in the development of stress-induced autophagy via tubulin acetylation. For this purpose Arabidopsis thaliana line expressing autophagy-related protein 8 h (atg8h)-GFP was generated to investigate autophagy, applying the level of free GFP as an indicator of autophagy development. Using autophagosome confocal imaging and Western blot analysis of Atg8 post-translational lipidation and synchronous GFP release it was shown that all examined stressful stimuli led to pronounced development of autophagy, particularly in different root tissues. Moreover, autophagy development was accompanied by alpha-tubulin acetylation under all stressful conditions. Presented data indicate the possible role of the post-translational acetylation of alpha-tubulin in the mediation of plant stress-induced autophagy

Genome-wide identification and analysis of cytokinin oxidase/dehydrogenase (ckx) gene family in finger millet (Eleusine coracana)

Cytokinin dehydrogenase/oxidase (CKX) enzymes play a key role in regulation of cytokinin (CK) levels in plants, degrading the excess levels of this phytohormone. It was shown that CKX appears to an attractive target for genetic engineering, since silencing of these genes boosts accumulation of cytokinin in various tissues, thus promoting rapid increase in biomass accumulation and general plant productivity. Previously we have reported about similar effect in somaclonal lines of finger millet (Eleusine coracana), caused by downregulation of EcCKX1 and EcCKX2. However, CKX gene family consists of numerous representatives, especially in allopolyploid crop species, such as E. coracana. To the date, whole CKX gene family has not been characterized for E. coracana and its close-related species. Here we first report about comprehensive genome-wide identification and analysis CKX gene panel in finger millet. Functional genes, identified within the genome of E. coracana, were compared with previously identified two genes EcCKX1 and EcCKX2. The exon-intron structure analysis, FAD- and CK-binding domains motif analysis were performed. Phylogeny of EcCKX genes was studied, suggesting that CKX genes are divided in several distinct groups, corresponding to specific isotypes. Finally, the phenotypic effect of EcCKX1 and EcCKX2 partial silencing in SE7 somaclonal line was investigated, suggesting that CKX-expression deficient lines demonstrate increased grain productivity, higher bushiness, enhanced biomass accumulation and shorter vegetation cycle