A. I. Yemets

Nitric oxide signalling via cytoskeleton in plants

Nitric oxide (NO) in plant cell mediates processes of growth and development starting from seed germination to pollination, as well as biotic and abiotic stress tolerance. However, proper understanding of the molecular mechanisms of NO signalling in plants has just begun to emerge. Accumulated evidence suggests that in eukaryotic cells NO regulates functions of proteins by their post-translational modifications, namely tyrosine nitration and S-nitrosylation. Among the candidates for NO-downstream effectors are cytoskeletal proteins because of their involvement in many processes regulated by NO. This review discusses new insights in plant NO signalling focused mainly on the involvement of cytoskeleton components into NO-cascades. Herein, examples of NO-related post-translational modifications of cytoskeletal proteins, and also indirect NO impact, are discussed. Special attention is paid to plant α-tubulin tyrosine nitration as an emerging topic in plant NO research.

Structural modeling of the interaction of plant α-tubulin with dinitroaniline and phosphoroamidate herbicides

Being the primary components of microtubules, tubulins are directly involved in fundamental processes, such as the spatial organization of subcellular components, dynamic distribution of intracellular compartments, and the establishment and maintenance of the form of eukaryotic cells (Mandelkow and Mandelkowa, 1995; Nogales, 1999). The function and activity of tubulin depend on its subcellular localization; however, recent efforts towards a more complete understanding have come from studies of the three-dimensional (3-D) structure of tubulin molecules (Nogales, 2000). Progress in these structural studies relies heavily on the use of 2-D crystalline sheets of animal tubulin (Nogales et al., 1995). The existence of a broad spectrum of information on tubulin sequences from different organisms, and the recent availability of results from crystallographic studies (Amos, 2000; Nogales et al., 1998), highlight the advantages of computer modeling. The spatial structure of this protein family has been studied (Gogonea et al., 1999), including plant tubulins (Nyporko and Blume, 2001, 2002), and has been shown to predict functionally significant features of tubulin molecules. The importance and necessity of acquiring this information are explained by following factors. For instance, the mechanism of action of a variety of compounds with important applied significance (e.g. herbicides, fungicides, anticancer and anti-helminthic drugs) is related to the ability of these agents to interact directly with tubulin molecules (Morejohn and Fosket, 1991; Strashnyuk and Blume, 1993). Among the low-molecular weight tubulin ligands, the dinitroaniline, phosphoroamidate and N-phenyl carbamate herbicides are compounds with a high specificity for plant tubulins than for animal tubulins (Antony and Hussey, 1999; Baird et al., 2000; Breviario and Nick, 2000; Yemets and Blume, 1999). Biochemical and molecular genetic investigation of the most thoroughly studied plant with natural resistance to dinitroanilines, a goosegrass (Eleusine indica (L.) Gaertn.), has shown that this resistance is caused by a point mutation at position 239 of the 1-tubulin molecule, which results in the replacement of the amino acid Thr by Ile (Antony et al., 1998; Yamamoto et al., 1998). This dinitroanilineresistant mutant exhibits cross-resistance to phosphoroamidates (Vaughn et al., 1987), and it was proposed earlier that these two chemically distinct classes of * Corresponding author. Tel./fax: +380-44-266-7104. E-mail address: alyemets@univ.kiev.ua (Ya.B. Blume).

Effects of tyrosine kinase and phosphatase inhibitors on microtubules in Arabidopsis root cells

To investigate the role of tyrosine phosphorylation/dephosphorylation processes in plant cells the morphology of Arabidopsis thaliana primary roots and the organization of cortical microtubules (MTs) were studied after inhibition of protein tyrosine kinases (PTKs) and tyrosine phosphatases (PTPs). It was found that all tested types of PTKs inhibitors (herbimycin A, genistein and tyrphostin AG 18) altered root hair growth and development, probably as a result of their significant influences on MTs organization in root hairs. The treatment also led to MTs reorientation and disruption in epidermis and cortex cells of both elongation and differentiation zones of primary roots. Enhanced tyrosine phosphorylation after treatment with a PTPs inhibitor (sodium orthovanadate) resulted in intense induction of root hair development and growth and caused a significant shortening of the elongation zone. It also led to changes of MTs orientation from transverse to longitudinal in epidermis and cortex cells of the elongation and differentiation zones of the root. From the data obtained we can suppose that tyrosine phosphorylation can be involved in the dynamics and organization of MTs in different types of plant cells.

Tyrosine phosphorylation of plant tubulin

Phosphorylation of αβ-tubulins dimers by protein tyrosine kinases plays an important role in the regulation of cellular growth and differentiation in animal cells. In plants, however, the role of tubulin tyrosine phosphorylation is unknown and data on this tubulin modification are limited. In this study, we used an immunochemical approach to demonstrate that tubulin isolated by both immunoprecipitation and DEAE-chromatography is phosphorylated on tyrosine residues in cultured cells of Nicotiana tabacum. This opens up the possibility that tyrosine phosphorylation of tubulin could be involved in modulating the properties of plant microtubules.

Functional role of nitric oxide in plants

The review considers involvement of nitric oxide (NO) in regulation of basic physiological processes underlying growth, development, and senescence in plants. The NO sources in plants, as well as direct and indirect NO signaling mechanisms are also reviewed. Particular attention is paid to the role of this secondary messenger in plant responses to various abiotic stresses, such as mechanical injury, salinity, drought, UV irradiation, high and low temperatures, ozonation, hypoxia, the impacts of heavy metals and herbicides. The role of NO in the hypersensitive response and in a systemic response upon plant infection with invasive pathogens is described.

Nitric oxide as a critical factor for perception of UV‐B irradiation by microtubules in Arabidopsis

Influence of ultraviolet-B (UV-B) as an abiotic stress factor on plant microtubules (MTs) and involvement of nitric oxide (NO) as a secondary messenger mediating plant cell response to environmental stimuli were investigated in this study. Taking into account that endogenous NO content in plant cells has been shown to be increased under a broad range of abiotic stress factors, the effects of UV-B irradiation and also the combined action of UV-B and NO donor sodium nitroprusside (SNP) or NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) on the MTs organization in different root cells of Arabidopsis thaliana were tested. Subsequently, realization of the MT-mediated processes such as root growth and development was studied under these conditions. Arabidopsis thaliana seedlings expressing the chimeric gene gfp-map4 were exposed to the enhanced UV-B with or without SNP or c-PTIO pretreatment. The UV-B irradiation alone led to a dose-dependent root growth inhibition and to morphological alterations of the primary root manifested in their swelling and excessive root hair formation. Moreover, dose-dependent randomization and depolymerization of MTs in both epidermal and cortical cells under the enhanced UV-B were found. However, SNP pretreatment of the UV-B irradiated A. thaliana seedlings recovered the UV-B inhibited root growth as compared to c-PTIO pretreatment. It has been shown that in 24 h after UV-B irradiation the organization of MTs in root epidermal cells of SNP-pretreated A. thaliana seedlings was partially recovered, whereas in c-PTIO-pretreated ones the organization of MTs has not been distinctly improved. Therefore, we suppose that the enhanced NO levels in plant cells can protect MTs organization as well as MT-related processes of root growth and development against disrupting effects of UV-B.

Development of transformation vectors based upon a modified plant α-tubulin gene as the selectable marker

A plant transformation and selection system has been developed utilizing a modified tubulin gene as a selectable marker. The vector constructs carrying a mutant α‐tubulin gene from goosegrass conferring resistance to dinitroaniline herbicides were created for transformation of monocotyledonous and dicotyledonous plants. These constructs contained β‐ and/or mutant α‐tubulin genes driven either by ubiquitin or CaMV 35S promoter. The constructs were used for biolistic transformation of finger millet and soybean or for Agrobacterium‐mediated transformation of flax and tobacco. Trifluralin, the main representative of dinitroaniline herbicides, was used as a selective agent in experiments to select transgenic cells, tissues and plantlets. Selective concentrations of trifluralin estimated for each species were as follows: 10 μM for Eleusine coracana, Glycine max, Nicotiana plumbaginifolia and Nicotiana sylvestris; 3 μM for Linum usitatissimum. PCR and Southern blotting analyses of transformed lines with a specific probe to nptII, α‐tubulin or β‐tubulin genes were performed to confirm the transgenic nature of regenerated plants. Band specific for the mutant α‐tubulin gene was identified in transformed plant lines. Results confirmed the stable integration of the mutant tubulin gene into the plant genomes. The present study clearly demonstrates the use of a plant mutant tubulin as a selective gene for plant transformation.

Tubulin tyrosine nitration regulates microtubule organization in plant cells

During last years, selective tyrosine nitration of plant proteins gains importance as well-recognized pathway of direct nitric oxide (NO) signal transduction. Plant microtubules are one of the intracellular signaling targets for NO, however, the molecular mechanisms of NO signal transduction with the involvement of cytoskeletal proteins remain to be elucidated. Since biochemical evidence of plant α-tubulin tyrosine nitration has been obtained recently, potential role of this posttranslational modification in regulation of microtubules organization in plant cell is estimated in current paper. It was shown that 3-nitrotyrosine (3-NO2-Tyr) induced partially reversible Arabidopsis primary root growth inhibition, alterations of root hairs morphology and organization of microtubules in root cells. It was also revealed that 3-NO2-Tyr intensively decorates such highly dynamic microtubular arrays as preprophase bands, mitotic spindles and phragmoplasts of Nicotiana tabacum Bright Yellow-2 (BY-2) cells under physiological conditions. Moreover, 3D models of the mitotic kinesin-8 complexes with the tail of detyrosinated, tyrosinated and tyrosine nitrated α-tubulin (on C-terminal Tyr 450 residue) from Arabidopsis were reconstructed in silico to investigate the potential influence of tubulin nitrotyrosination on the molecular dynamics of α-tubulin and kinesin-8 interaction. Generally, presented data suggest that plant α-tubulin tyrosine nitration can be considered as its common posttranslational modification, the direct mechanism of NO signal transduction with the participation of microtubules under physiological conditions and one of the hallmarks of the increased microtubule dynamics.

Progress in Plant Polyploidization Based on Antimicrotubular Drugs

The results of artificial polyploidization for horticultural and agronomic plant species are presented in this re- view. The data on use of the classical antimitotic drug colchicine for chromosome doubling are reported. The efficacy of other compounds such as dinitroanilines and phosphorothioamidates in polyploidy induction as compared to colchicine has been summarized. The molecular basis of highly specific binding of dinitroanilines and phosphorothioamidates with plant tubulin for induction of efficient polyploidy are discussed.

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

BackgroundThe 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.ResultsUsing homology modeling we have generated an Arabidopsis thaliana 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.ConclusionsWe predicted surface exposure of five β-tubulin epitopes, as well as tyrosine residues, on the surface of A. thaliana microtubule protofilament model, and validated the obtained results by immunofluorescence microscopy on cortical microtubules in cells.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.