Ya. B. Blume

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).

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.

Heavy metals have a different action from aluminium in disrupting microtubules in Allium cepa meristematic cells

In our previous investigations (Dovgalyuk et al., 2001), phytotoxic metals such as Cd, Pb, Ni and Al were shown to disturb mitotic and cytokinetic processes in onion meristematic cells. Very likely those effects resulted from damage of microtubules of the mitotic spindle and phragmoplast. To verify this assumption, indirect immunocytochemical analysis of microtubule structures was conducted in the root tip cells pretreated for 24 h with 50 μM CdCl2, 50 μM Pb(CH3COO)2, 100 μM NiSO4 and 100 μM Al(NO3)3 (salt concentrations which caused the highest effects). The results demonstrated that different metals had different effects on microtubular organization. In controls, cells contained a highly organized intricate network of numerous microtubules that was characterized by a slightly wavy curving morphology. Such cells were able to form four different types of structures depending on the cell cycle stage: the interphase array, preprophase band, mitotic spindle and phragmoplast. Exposure of onion cells to 50 μM CdCl2 resulted in profound disturbance of microtubule organization resulting from severe disassembly of microtubules evidenced by numerous short microtubule fragments at the cell periphery (Fig. 1). Similar effects were shown in Swiss 3T3 mouse cells exposed to CdCl2 (Chou, 1989). Such abnormality of microtubule organization may have resulted from an inappropriate activation of calmodulin induced by cadmium ions (Perrino, 1989). Furthermore, cadmium chloride is an effective sulfhydryl binding compound and so it is able to reduce the amount of free sulfhydryl groups on tubulin, which are essential for microtubule polymerization (Wallin and Hartley-Asp, 1993). In our experiments Pb(CH3COO)2 caused a disorganization of microtubular structures in a manner similar to that of CdCl2. Small fragments only of microtubules have been observed at the nuclear surface and cell periphery, but a weaker antimicrotubular activity of Pb was revealed. Cells with an almost typical microtubule organization were present amongst cells with disarranged microtubules. Mechanisms of influence of this toxic metal on the microtubules in vitro and in living cells have not been sufficiently studied yet. Perhaps it is similar to that of Cd injury. Interestingly, a very different type of cytoskeletal perturbation was observed in cells exposed to NiSO4. Treatment with Ni resulted in the appearance of thick microtubular bundles or aggregates in the perinuclear region of interphase cells and their deficiency in the cell periphery (Fig. 1b). It was also found that microtubules of mitotic spindle were disturbed by Ni ions and fragments only of mitotic spindles were present (Fig. 1c). Earlier it was shown that Ni enhanced the in vitro polymerization of animal tubulin resulting in the formation of much shorter but more numerous microtubules (Lin and Chou, 1990). In contrast to these compounds, Al(NO3)3 did not break down microtubular structures, it resulted in more dense packing of microtubules in the cells (Fig. 1d). Such stabilizing action resembled that of taxol. More studies have been carried out on the influence of Al on plant and animal microtubules than for any other metals but the mechanism of its effects on plant microtubules remain insufficiently investigated. It is known that aluminium treatment promotes the in vitro assembly of tubulin in the absence of microtubule associated proteins, and inhibits subsequent Ca-induced depolymerization of microtubules (MacDonald et al., 1987). The same authors suggested that, in vitro, the Al ions might * Corresponding author. Tel.: +380-322-72-59-08; fax: +380-322-70-74-30. Cell Biology International 27 (2003) 193–195 Cell Biology International

Transfer of amiprophosmethyl resistance from a Nicotiana plumbaginifolia mutant by somatic hybridisation

Abstract Transfer of resistance to the phosphorothioamidate herbicide, amiprophosmethyl (APM), from the β-tubulin mutant of Nicotiana plumbaginifolia to the interspecific N. plumbaginifolia (+)N. sylvestris and to the intertribal N. plumbaginifolia (+) Atropa belladonna somatic hybrids has been demonstrated. Transfer to the recipient species was accomplished by: (1) symmetric hybridisationand (2) asymmetric hybridisation using γ-irradiation of donor protoplasts. Cytogenetic analysis confirmed the hybrid origin of the hybrids obtained. It was established that most of them typically inherited no more than three donor chromosomes, although it was possible to obtain symmetric hybrids in the case of symmetric fusion. Immunofluorescent microscopy analysis has shown that protoplasts of the mutant, and of the N. plumbaginifolia (+) N. sylvestris and N. plumbaginifolia (+) A. belladonna hybrids, retained the normal structure of interphase microtubule (MT) arrays and mitotic figures after treatment with 5 µM APM, whereas MTs of protoplasts of the recipients were destroyed under these conditions. It was also shown that hybrid clones contained an altered β-tubulin isoform originating from the N. plumbaginifolia mutant. The selected hybrid clones were characterised by cross-resistance to trifluralin, a dinitroaniline herbicide with the same mode of anti-MT action. Some of the somatic hybrids which could flower were fertile. It was established that seeds of some fertile hybrids were able to germinate in the presence of 5 µM APM. The results obtained thus support the conclusion that the technique of somatic hybridisation, especially asymmetric fusion, can be used to transfer APM resistance from the N. plumbaginifolia mutant to different (related and remote) plant species of the Solanaceae, including important crops.

Cold adaptation of plant microtubules: structural interpretation of primary sequence changes in a highly conserved region of α-tubulin

The microtubules of higher plants in particular are one of the cell components, participating in the cold stress response and in adaptation to low temperatures (Nick, 2000). Microtubules can play a dual role in the response of plants to the cold. First, they are depolymerized after exposure to low positive temperatures—a phenomenon correlating with changes of plant cell growth (Pihakaski-Maunsbach and Puhakainen, 1995). Such microtubule depolymerization triggers a cascade of cellular processes, that results in general plant reactions to cold (Durso and Cyr, 1994; Mazars et al., 1997). A second function is to increase cold stability. This is seen in a number of organisms: animals (Antarctic fishes; Detrich, 1997; Detrich et al., 2000), fungi (Gupta et al., 2001) and psychrophilic algae (Willem et al., 1999) and clearly correlates with the cold-resistance of their microtubules. Such resistance is explained by amino acid substitutions in sequences of tubulin molecules, specific for each kingdom. Comparative analysis of different sequences of tubulin isotypes from organisms with different degrees of cold-resistance can allow the detection of tubulin structural domains responsible for microtubular adaptation to low temperatures. The comparative analysis of -tubulin sequences of the psychrophilic alga Chloromonas and the mesophilic alga Chlamydomonas reinhardtii has shown that they are characterized by a few very interesting amino acid differences, which could be considered as reliable candidates for explaining the increased cold-resistance of psychrophilic algae (Willem et al., 1999). Such differences include substitution of Met268 on Val in Chloromonas -tubulin. Very interestingly, this position is located in a highly conservative region of the tubulin molecule (Anthony and Hussey, 1999) and its replacement by a Thr correlates genetically with intermediate dinitroaniline-resistant phenotypes of goosegrass (Eleusine indica; Yamamoto et al., 1998). Development of tools for modelling the spatial structure of tubulin (Nogales, 2000), and their adaptation for plant tubulins (Nyporko and Blume, 2001), provides a possibility to estimate the influence of these specific replacements on spatial features of tubulin molecules. To realize this possibility we developed spatial models of tubulin molecules from the psychrophilic alga Chloromonas and the mesophilic alga Ch. reinhardtii on the basis of their primary structure data. We also developed a spatial model of goosegrass -tubulin with a Chloromonas specific replacement at position 268—the most reliable candidate for induction of microtubule cold-resistance. The models were compared with each other and with a model of E. indica -tubulin with the aim of showing the significance of this replacement for the regulation of tubulin dimer–dimer interaction.

Sensitivity of Eleusine indica Callus to Trifluralin and Amiprophosmethyl in Correlation with the Binding of These Compounds to Tubulin

The sensitivity of calluses derived from susceptible and resistant goosegrass (Eleusine indica (L.) Gaertn.) biotypes to dinitroaniline herbicides, which disrupt interphase and mitotic-spindle microtubules, was evaluated. A callus culture derived from the resistant biotype retained resistance to both trifluralin (dinitroaniline herbicide) and amiprophosmethyl (phosphorothioamidate herbicide). The site for the interaction between α-tubulin subunit and dinitroaniline or phosphorothioamidate herbicides was identified by computer simulation. A correlation was found between the level of callus sensitivity to herbicide tested and the pattern of herbicide interaction with α-tubulin.

Microtubule reorganization as a response to implementation of NO signals in plant cells

The effects of an exogeneous NO donor, sodium nitroprusside, on the orientation and organization of cortical microtubules in Arabidopsis thaliana root cells expressing GFP-MAP4 were studied in vivo. It was found that sodium nitroprusside treatment (10–500 μM, 24 h) caused the acceleration of primary root growth and enhanced initiation of root hairs in the differentiation zone. The influence of sodium nitroprusside revealed in changes in the orientation and organization of cortical microtubules in different types of cells of A. thaliana root. The most sensitive to sodium nitroprusside exposure were microtubules in epidermal cells of the elongation zone, where native transverse orientation of cortical microtubules turned into random, oblique, or longitudinal relative to the primary root axis. We suggest that NO, as one of the intracellular secondary messengers, triggers cell differentiation by reorientation of cortical microtubules, possibly via tubulin nitrotyrosination.

Alteration of β-tubulin in Nicotiana plumbaginifolia confers resistance to amiprophos-methyl

Abstract A Nicotiana plumbaginifolia plant (apm5r) resistant to amiprophos-methyl (APM), a phosphoro-amide herbicide, was isolated from protoplasts prepared from leaves of haploid plants. Genetic analysis revealed that the resistance is coded for by a dominant nuclear mutation and is associated with the increased stability of cortical microtubules. Two-dimensional polyacrylamide-gel electrophoresis, combined with immunoblotting using anti-tubulin monoclonal antibodies, showed that part of the β-tubulin in the resistant plant possessed lower isoelectric points than the β-tubulin of susceptible wild-type plants. These results provide evidence that the resistance to APM is associated with a mutation in a β-tubulin gene. The APM-resistant line showed cross-resistance to trifluralin, a dinitroaniline herbicide, suggesting a common mechanism of resistance between these two classes of herbicides.

Effects of phytohormones on the cytoskeleton of the plant cell

This review highlights the effects of “classic” phytohormones (auxins, cytokinins, gibberellins, abscisic acid, ethylene, and brassinosteroids) and also of important signaling molecules, such as jasmonic acid, strigolactones, and nitric oxide, on the main components of the plant cytoskeleton, microtubules and microfilaments. The effects of these growth regulators on orientation and organization of microtubules and actin filaments, realization of cytoskeleton-dependent processes, expression of tubulin and actin genes, and interaction of various phytohormones in their influence on the cytoskeleton are discussed.

Inhibitors of tyrosine kinases and phosphatases as a tool for the investigation of microtubule role in plant cold response

Tyrosine phosphorylation plays a vital role in the variety of signal transduction pathways in eukaryotic cells, however its role and relevance in plants are still largely unknown. To investigate the functional role of tubulin tyrosine phosphorylation in plant cells the interplay between the effects of tyrosine kinases (herbimycin A) as well as tyrosine phosphatases (sodium orthova nadate) inhibitors on microtubules sensitivity to cold in A. thaliana root cells were studied. Since it was found that inhibition of tyrosine kinases significantly increased the microtubules sensitivity to cold, while inhibition of tyrosine phophatases enhanced their cold-resistance, we suggest an existence of certain functional interaction between the phosphorylation on tyrosine residues and sensitivity of cortical microtubules to low temperatures.