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Cortical microtubule orientation in Arabidopsis thaliana root meristematic zone depends on cell division and requires severing by katanin
© The Author(s) 2018
Received: 29 January 2018
Accepted: 8 June 2018
Published: 15 June 2018
Transverse cortical microtubule orientation, critical for anisotropic cell expansion, is established in the meristematic root zone. Intending to elucidate the possible prerequisites for this establishment and factors that are involved, microtubule organization was studied in roots of Arabidopsis thaliana, wild-type and the p60-katanin mutants fra2, ktn1-2 and lue1. Transverse cortical microtubule orientation in the meristematic root zone has proven to persist under several regimes inhibiting root elongation. This persistence was attributed to the constant moderate elongation of meristematic cells, prior to mitotic division. Therefore, A. thaliana wild-type seedlings were treated with aphidicolin, in order to prevent mitosis and inhibit premitotic cell elongation.
In roots treated with aphidicolin for 12 h, cell divisions still occurred and microtubules were transverse. After 24 and 48 h of treatment, meristematic cell divisions and the prerequisite elongation ceased, while microtubule orientation became random. In meristematic cells of the p60-katanin mutants, apart from a general transverse microtubule pattern, cortical microtubules with random orientation were observed, also converging at several cortical sites, in contrast to the uniform transverse pattern of wild-type cells.
Taken together, these observations reveal that transverse cortical microtubule orientation in the meristematic zone of A. thaliana root is cell division-dependent and requires severing by katanin.
Axial growth of plant root is achieved by cell proliferation and elongation. New cells are produced in the meristematic zone, prepare for rapid expansion in the transition zone and finally elongate in the fast elongation zone . In all the developmental root zones, anisotropic cell expansion requires transversely oriented cellulose microfibrils in the cell wall, following the arrangement of cortical microtubules [2–5]. Recently, it has been confirmed in Arabidopsis thaliana that transverse cortical microtubule orientation is initially established in the meristematic root zone [4, 5]. This orientation is “bequeathed” to the transition and fast elongation zones, as long as cells expand anisotropically parallel to the root axis. In addition, transverse microtubule orientation appears more persistent in the meristematic zone than in the fast elongation zone under experimental conditions that inhibit elongation [4, 5].
To interpret this difference in cortical microtubule orientation persistence, it was suggested that neither genetic nor chemically-induced inhibition of cellulose synthesis and/or conformation may disrupt the cell cycle in meristematic cells. Accordingly, cell expansion prior to cell division perseveres and is a critical factor for establishing and maintaining transverse microtubule orientation in meristematic root cells [4, 5].
In the present study, this hypothesis was challenged. Cortical microtubule organization was studied in wild-type A. thaliana primary roots, in which cells stopped dividing after treatment with aphidicolin, a potent inhibitor of DNA replication . In addition, in order to further investigate the factors involved in microtubule orientation, we compared cortical microtubule arrangement in meristematic root cells of the wild-type and of the p60-katanin mutants fra2 , lue1  and ktn1/2 . Our findings support that establishment of transverse cortical microtubule orientation in the meristematic zone of A. thaliana root is substantially associated to cell division and depends on microtubule severing.
Seeds of A. thaliana, wild-type (Col-0) and the p60-katanin mutants fra2, lue1 and ktn1/2, were surface sterilized and grown on solid agar medium as previously described . All the chemicals and reagents used in this study were purchased from Sigma (Taufkirchen, Germany), Merck (Darmstadt, Germany) and Applichem (Darmstadt, Germany), and all the following steps were carried out at room temperature unless stated otherwise.
Five-day-old wild-type seedlings were transplanted on solid agar medium plates supplemented with 30 μM aphidicolin and further grown for 12, 24 or 48 h. Untreated and aphidicolin-treated wild-type seedlings, as well as untreated mutant seedlings, were prepared for whole-mount α-tubulin immunolabeling as follows: whole seedlings were fixed for 1 h in 4% (w/v) paraformaldehyde in PEM (50 mM PIPES, 5 mM EGTA, 5 mM MgSO4, pH 6.8) with the addition of 5% (v/v) dimethylsulfoxide (DMSO). After washing in PEM (3 × 10 min), cell walls were digested for 1 h in 2% (w/v) macerozyme R-10 (Duchefa, Haarlem, Netherlands) in PEM. Then, the seedlings were treated with absolute methanol at − 20 °C for 20 min and subsequently extracted with 5% (v/v) DMSO and 1% (v/v) Triton X-100 for 1 h. Incubations with rat anti-α-tubulin (YOL 1/34, AbD Serotec, Kidlington, UK) and FITC-anti-rat (Invitrogen, Carlsbad, CA), both diluted 1:40 in PEM, were carried out sequentially overnight in the dark with a washing intermediate step (3 × 10 min). Finally, after washing in PEM as previously, the seedlings were mounted in a PEM-glycerol mixture (1:2 v/v) supplemented with 0.5% p-phenylenediamine as anti-fade agent. For each treatment, 10 primary roots were studied. Some specimens were slightly squashed between the microscope slide and coverslip, to release the cortex cells from the surrounding tissues. The preparations were examined with a Nikon D-Eclipse C1 or a Zeiss LSM780 confocal laser scanning microscope (CLSM), with the appropriate filters for FITC, and micrographs were acquired with each manufacturer’s software.
In the images obtained with the above CLSM systems, angular distribution of cortical microtubules was analyzed and deciphered using the Microfilament Analyzer software (https://www.uantwerpen.be/en/research-groups/bimef/downloads/microfilament-analyzer/). By means of this software the angular distribution of cortical microtubules could be determined and accordingly inferred to their orientation either as “transverse”, at angles of 90° or 270°, “longitudinal” at angles of 0° or 180° or “random” at angles between 0° and 180° as described in . Statistical analysis (ANOVA with Dunnett’s multiple comparison test) of cell length in untreated and aphidicolin-treated roots was performed with GraphPad (San Diego, CA, USA), with significance at p < 0.05.
Roots of untreated wild-type, fra2, lue1 and ktn1/2 seedlings were also prepared for transmission electron microscopy (TEM) as previously described . In brief, root segments comprising the developmental root zones were fixed for 4 h in 3% (v/v) glutaraldehyde in 50 mM sodium cacodylate, pH 7, post-fixed in 1% (w/v) osmium tetroxide for 3 h, dehydrated in an acetone series and embedded in Spurr’s resin. Ultrathin sections (70–90 nm) were double stained with uranyl acetate and lead citrate and observed with a JEOL JEM 1011 TEM. Images were acquired with a Gatan ES500 W camera. Confocal and TEM images were processed with Adobe Photoshop CS2 with only linear settings.
Results and discussion
Number and percentage of cells at various cell cycle stages, in the meristematic root zone of control and aphidicolin-treated roots
After 24 h of aphidicolin treatment, cell divisions had completely ceased (Table 1) in all the roots that were studied. Cortical microtubules exhibited random orientation in the cells of the meristematic zone (Fig. 1c). The above finding was further confirmed using the Microfilament Analyzer software. It appears that randomly oriented cortical microtubules were amplified over the transverse ones, as aphidicolin treatment duration increased (Fig. 1e–h).
After 48 h of treatment, apart from cessation of cell divisions (Table 1), the cells of the meristematic zone appeared vacuolated and exhibited randomly oriented cortical microtubules (Fig. 1d), while cell length spanned between 9 and 20 μm (Fig. 2a). Vacuolation is typical of meristematic cells under stress [14, 15] and its occurrence can be considered as a side-effect of aphidicolin, which at longer treatment becomes fatal. Elongated nuclei like those reported by Yasuhara and Kitamoto  were not observed. However, this may be due to the difference between the cell type studied there (tobacco BY2 cells) and A. thaliana root cells.
Cell size is a critical factor for the progression of plant cell cycle . Plant cells grow to achieve a size large enough to be divided in two cells, which in turn will grow to divide again. In the root of A. thaliana the majority of cell divisions are proliferative, perpendicular to the root axis, especially over the T-divisions . As a result, meristematic cell growth is mainly anisotropic, parallel to the root axis. This moderate expansion, though overlooked , is necessary for cell proliferation in the meristematic root zone. Our data provide evidence that the bidirectional interplay between cell expansion and cortical microtubule arrangement, previously shown in fast elongating cells [4, 5], also exists in the meristematic root zone: anisotropic cell expansion is sustained by transversely arranged microtubules, while the latter arrangement requires cell expansion to be established and maintained. Overall, initiation, establishment and maintenance of transverse microtubule orientation in the developmental zones of A. thaliana root depend on cell division and require severing by p60-katanin.
EP designed the experiments, obtained CLSM and TEM data, analyzed the results and wrote the manuscript. I-DSA performed the experiments, obtained and analyzed CLSM data and contributed to manuscript preparation. B-ED obtained and analyzed TEM data and contributed to manuscript preparation. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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This work was financially supported by the Research Committee of Aristotle University of Thessaloniki (Grant Nos. 89348 and 91913).
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