doi: 10.15389/agrobiology.2018.3.634eng

UDC 631.461.52:576:576.086

The work was carried out on the equipment of the ARRIAM Center for Genomic Technologies, Proteomics and Cell Biology and of the BIN RAS Center for Cell and Molecular Technologies of Studying Plants and Fungi.
Supported financially by Russian Science Foundation (grant 16-16-10035)



A.B. Kitaeva1, P.G. Kusakin1, K.N. Demchenko1,2, V.E. Tsyganov1

1All-Russian Research Institute for Agricultural Microbiology, Federal Agency of Scientific Organizations, 3, sh. Podbel’skogo, St. Petersburg, 196608 Russia, (✉ corresponding author);
2Komarov Botanical Institute RAS, Federal Agency of Scientific Organizations, 2, ul. Professora Popova, St. Petersburg, 197376 Russia, e-mail

Kitaeva A.B.
Demchenko K.N.
Kusakin P.G.
Tsyganov V.E.

Received November 29, 2017


The discovery of microtubules in plants, as well as their subsequent study, was made possible by the methods of electron microscopy. Further, methods for visualizing the cytoskeleton in a plant cell were actively developed using immunolocalization combined with laser scanning confocal microscopy (K. Celler et al., 2016). All the above-listed methods involve the fixation of the analyzed biological material. It should be noted that the tubulin cytoskeleton is an extremely dynamic structure; therefore, techniques of microtubule visualization in living plant cells using fluorescent proteins have been actively developed in recent years (K. Celler et al., 2016). Nevertheless, immunohistochemical analysis is still an essential method (J. Dyachok et al., 2016). First of all, this is due to the fact that in vivo observations are limited to plant cells of the surface layers (root hairs, epidermis) (F.M. Perrine-Walker et al., 2014; J. Dyachok et al., 2016). Moreover, for many plant species, the size of their organs is much larger than that of Arabidopsis thaliana, which makes it impossible to analyze changes in the organization of the cytoskeleton in vivo (J. Dyachok et al., 2016). Another limiting factor is that for several plant species, transformation protocols have not yet been developed or are very difficult, e.g., pea (Pisum sativum L.) (A. Iantcheva et al., 2013). In optimization of the protocols for effective immunohistochemical analysis of the tubulin cytoskeleton, the fixation of plant material is an important step. In our study, it was shown that this optimization is required when new legume species are studied. For instance, the protocol for pea nodule fixation developed by us required changes when applied to the nodules of Medicago truncatula. Moreover, modifications in the protocol for fixation may even be necessary when examining different mutants in the symbiotic genes of a plant species, because such mutations can exert a strong influence on the physicochemical properties of the nodule tissues. Therefore, we used various fixation protocols for the wild-type line of M. truncatula A17 and its mutants dnf1-1, efd-1 and TR3 (ipd3). It has also been shown that the preparation of sections of fixed nodules using a microtome with a vibrating blade can significantly improve the preservation of the structure of the tubulin cytoskeleton as compared to the use of fixed specimens embedded in Steedman’s wax and subsequent sectioning using a rotary microtome. It was found that the age of the nodules is also an important factor in the visualization of the tubulin cytoskeleton. To compare the patterns of tubulin cytoskeleton in different cell types, quantitative analysis is required. We found that the MicroFilament Analyzer (E. Jacques et al., 2013) with additional scripts seemed well suited for checking the frequency of microtubules with a given orientation.

Keywords: legume-rhizobial symbiosis, microtubules, immunolocalization, Pisum sativum, Medicago truncatula, quantitative analysis, MicroFilament Analyzer.


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