doi: 10.15389/agrobiology.2018.3.616eng

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.


Full article (Rus)

Full article (Eng)



  1. Celler K., Fujita M., Kawamura E., Ambrose C., Herburger K., Holzinger A., Wasteneys G.O. Microtubules in plant cells: strategies and methods for immunofluorescence, transmission electron microscopy, and live cell imaging. In: Cytoskeleton methods and protocols. Methods in molecular biology. R.H. Gavin (ed.). Humana Press, New York, NY, 2016, V. 1365: 155-184 CrossRef
  2. Dyachok J., Paez-Garcia A., Yoo C.M., Palanichelvam K., Blancaflor E.B. Fluorescence imaging of the cytoskeleton in plant roots. In: Cytoskeleton methods and protocols: methods and protocols In: Cytoskeleton methods and protocols. Methods in molecular biology. R.H. Gavin (ed.). Humana Press, New York, NY, 2016, V. 1365: 139-153 CrossRef
  3. Iantcheva A., Mysore K.S., Ratet P. Transformation of leguminous plants to study symbiotic interactions. Int. J. Dev. Biol., 2013, 57(6-8): 577-586 CrossRef
  4. Collings D.A., Wasteneys G.O. Actin microfilament and microtubule distribution patterns in the expanding root of Arabidopsis thaliana. Can. J. Bot., 2005, 83(6): 579-590 CrossRef
  5. Bakhuizen R. The plant cytoskeleton in the Rhizobium-Legume symbiosis. Ph.D. Dissertation. Leiden, The Netherlands: Leiden University, 1988.
  6. Timmers A.C.J., Auriac M.-C., Truchet G. Refined analysis of early symbiotic steps of the Rhizobium-Medicago interaction in relationship with microtubular cytoskeleton rearrangements. Development, 1999,126(16): 3617-3628.
  7. Sieberer B.J., Timmers A.C.J., Emons A.M.C. Nod factors alter the microtubule cytoskeleton in Medicago truncatula root hairs to allow root hair orientation. Mol. Plant-Microbe Interact., 2005, 18(11): 1195-1204 CrossRef
  8. Vassileva V.N., Kouchi H., Ridge R.W. Microtubule dynamics in living root hairs: transient slowing by lipochitin oligosaccharide nodulation signals. Plant Cell, 2005, 17(6): 1777-1787 CrossRef
  9. Timmers A.C.J., Vallotton P., Heym C., Menzel D. Microtubule dynamics in root hairs of Medicago truncatula. Eur. J. Cell Biol., 2007, 86(2): 69-83 CrossRef
  10. Perrine-Walker F.M., Lartaud M., Kouchi H., Ridge R.W. Microtubule array formation during root hair infection thread initiation and elongation in the Mesorhizobium-Lotus symbiosis. Protoplasma, 2014, 251(5): 1099-1111 CrossRef
  11. Timmers A.C.J., Auriac M.-C., de Billy F., Truchet G. Nod factor internalization and microtubular cytoskeleton changes occur concomitantly during nodule differentiation in alfalfa. Development, 1998, 125(3): 339-349.
  12. Whitehead L.F., Day D.A., Hardham A.R. Cytoskeleton arrays in the cells of soybean root nodules: the role of actin microfilaments in the organisation of symbiosomes. Protoplasma, 1998, 203(3-4): 194-205 CrossRef
  13. Davidson A.L., Newcomb W. Organization of microtubules in developing pea root nodule cells. Can. J. Bot., 2001, 79(7): 777-786 CrossRef
  14. Fedorova E.E., de Felipe M.R., Pueyo J.J., Lucas M. Conformation of cytoskeletal elements during the division of infected Lupinus albus L. nodule cells. J. Exp. Bot., 2007, 58(8): 2225-2236 CrossRef
  15. Timmers A.C.J. The role of the plant cytoskeleton in the interaction between legumes and rhizobia. Journal of Microscopy, 2008, 231(2): 247-256 CrossRef
  16. Norenburg J.L., Barrett J.M. Steedman’s polyester wax embedment and de-embedment for combined light and scanning electron microscopy. J. Elec. Microsc. Tech., 1987, 6(1): 35-41 CrossRef
  17. Baluška F., Parker J.S., Barlow P.W. Specific patterns of cortical and endoplasmic microtubules associated with cell growth and tissue differentiation in roots of maize (Zea mays L.). J. Cell Sci.,1992, 103(1): 191-200.
  18. Kitaeva A.B., Demchenko K.N., Tikhonovich I.A., Timmers A.C.J., Tsyganov V.E. Comparative analysis of the tubulin cytoskeleton organization in nodules of Medicago truncatula and Pisum sativum: bacterial release and bacteroid positioning correlate with characteristic microtubule rearrangements. New Phytol., 2016, 210(1): 168-183 CrossRef
  19. Stumpe M., Göbel C., Demchenko K., Hoffmann M., Klösgen R.B., Pawlowski K., Feussner I. Identification of an allene oxide synthase (CYP74C) that leads to formation of α-ketols from 9-hydroperoxides of linoleic and linolenic acid in below-ground organs of potato. Plant J., 2006, 47(6): 883-896 CrossRef
  20. Kosterin O.E., Rozov S.M. Mapping of the new mutation blb and the problem of integrity of linkage group I. Pisum Genetics, 1993, 25: 27-31.
  21. Berdnikov V.A., Rozov S.M., Bogdanova B.C. Materialy konferentsii «Chastnaya genetika rastenii» (23-25 maya 1989 goda, g. Kiev) [Proc. Conf. «Plant genetics», May 23-25, 1989, Kiev, Ukraine]. Kiev, 1989, V. 2: 47-51 (in Russ.).
  22. Tsyganov V.E., Morzhina E.V., Stefanov S.Y., Borisov A.Y., Lebsky V.K., Tikhonovich I.A. The pea (Pisum sativum L.) genes sym33 and sym40 control infection thread formation and root nodule function. Mol. Gen. Genet., 1998, 256(5): 491-503 CrossRef
  23. Borisov A.Y., Rozov S.M., Tsyganov V.E., Kulikova O.A., Kolycheva A.N., Yakobi, L.M., Ovtsyna A.O., Tikhonovich I.A. Identification of symbiotic genes in pea (Pisum sativum L.) by means of experimental mutagenesis. Russ. J. Genet., 1994, 30(11): 1284-1292.
  24. Wang D., Griffitts J., Starker C., Fedorova E., Limpens E., Ivanov S., Bisseling T., Long S. Nodule-specific protein secretory pathway required for nitrogen-fixing symbiosis. Science, 2010, 327(5969): 1126-1129 CrossRef
  25. Vernié T., Moreau S., De Billy F., Plet J., Combier J.P., Rogers C., Oldroyd G., Frugier F., Niebel A., Gamas P. EFD is an ERF transcription factor involved in the control of nodule number and differentiation in Medicago truncatula. Plant Cell, 2008,20(10): 2696-2713 CrossRef
  26. Maunoury N., Redondo-Nieto M., Bourcy M., Van de Velde W., Alunni B., Laporte P., Durand P., Agier N., Marisa L., Vaubert D., Delacroix H., Duc G., Ratet P., Aggerbeck L., Kondorosi E., Mergaert P. Differentiation of symbiotic cells and endosymbionts in Medicago truncatula nodulation are coupled to two transcriptome-switches. PLoS ONE, 2010, 5(3): e9519 CrossRef
  27. Ovchinnikova E., Journet E.-P., Chabaud M., Cosson V., Ratet P., Duc G., Fedorova E., Liu W., den Camp R.O., Zhukov V., Tikhonovich I., Borisov A., Bisseling T., Limpens E. IPD3 controls the formation of nitrogen-fixing symbiosomes in pea and Medicago spp. Mol. Plant-Microbe Interact., 2011, 24(11): 1333-1344 CrossRef
  28. Schindelin J., Arganda-Carreras I., Frise E., Kaynig,V., Longair M., Pietzsch T., Preibisch S., Rueden C., Saalfeld S., Schmid B., Tinevez J.Y., White D.J., Hartenstein V., Eliceiri K., Tomancak P., Cardona A. Fiji: an open-source platform for biological-image analysis. Nat. Methods, 2012, 9(7): 676-682 CrossRef
  29. Sampathkumar A., Lindeboom J.J., Debolt S., Gutierrez R., Ehrhardt D.W., Ketelaar T., Persson S. Live cell imaging reveals structural associations between the actin and microtubule cytoskeleton in Arabidopsis. Plant Cell, 2011, 23(6): 2302-2313 CrossRef
  30. Boudaoud A., Burian A., Borowska-Wykret D., Uyttewaal M., Wrzalik R., Kwiatkowska D., Hamant O. FibrilTool, an ImageJ plug-in to quantify fibrillar structures in raw microscopy images. Nat. Protoc., 2014, 9(2): 457-463 CrossRef
  31. Rezakhaniha R., Agianniotis A., Schrauwen J.T.C., Griffa A., Sage D., Bouten C.V.C., van de Vosse F.N., Unser M., Stergiopulos N. Experimental investigation of collagen waviness and orientation in the arterial adventitia using confocal laser scanning microscopy. Biomech. Model. Mechanobiol., 2012, 11(3-4): 461-473 CrossRef
  32. Jacques E., Buytaert, J., Wells D.M., Lewandowski M., Bennett M.J., Dirckx J., Verbelen J.P., Vissenberg K. MicroFilament Analyzer, an image analysis tool for quantifying fibrillar orientation, reveals changes in microtubule organization during gravitropism. Plant J., 2013, 74(6): 1045-1058 CrossRef
  33. Wickham H. ggplot2: Elegant graphics for data analysis. 2nd Edition. Springer International Publishing, 2016 CrossRef