The development of the root apex is determined by progress of cells from the meristematic region to the successive post-mitotic developmental zones for transition, cell elongation and final cell differentiation. We addressed root development, tissue architecture and root developmental zonation by means of light-sheet microscopic imaging of Arabidopsis thaliana seedlings expressing END BINDING protein 1c (EB1c) fused to green fluorescent protein (GFP) under control of native EB1c promoter. Unlike the other two members of the EB1 family, plant-specific EB1c shows prominent nuclear localization in non-dividing cells in all developmental zones of the root apex. The nuclear localization of EB1c was previously mentioned solely in meristematic cells, but not further addressed. With the help of advanced light-sheet microscopy, we report quantitative evaluations of developmentally-regulated nuclear levels of the EB1c protein tagged with GFP relatively to the nuclear size in diverse root tissues (epidermis, cortex, and endodermis) and root developmental zones (meristem, transition, and elongation zones). Our results demonstrate a high potential of light-sheet microscopy for 4D live imaging of fluorescently-labeled nuclei in complex samples such as developing roots, showing capacity to quantify parameters at deeper cell layers (e.g., endodermis) with minimal aberrations. The data presented herein further signify the unique role of developmental cell reprogramming in the transition from cell proliferation to cell differentiation in developing root apex.

Department of Cell Biology Centre of the Region Haná for Biotechnological and Agricultural Research Palacký University Olomouc Olomouc Czech Republic

Zobrazit více v PubMed

Adachi S., Minamisawa K., Okushima Y., Inagaki S., Yoshiyama K., Kondou Y., et al. . (2011). Programmed induction of endoreduplication by DNA double-strand breaks in Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. 108, 10004–10009. 10.1073/pnas.1103584108 PubMed DOI PMC

Akhmanova A., Steinmetz M. O. (2008). Tracking the ends: a dynamic protein network controls the fate of microtubule tips. Nat. Rev. Mol. Cell Biol. 9, 309–322. 10.1038/nrm2369 PubMed DOI

Baluška F., Kubica S., Hauskrecht M. (1990). Postmitotic' isodiametric' cell growth in the maize root apex. Planta 181, 269–274. 10.1007/BF00195876 PubMed DOI

Baluška F., Mancuso S. (2013). Root apex transition zone as oscillatory zone. Front. Plant Sci. 4:354. 10.3389/fpls.2013.00354 PubMed DOI PMC

Baluska F., Vitha S., Barlow P. W., Volkmann D. (1997). Rearrangements of F-actin arrays in growing cells of intact maize root apex tissues: a major developmental switch occurs in the postmitotic transition region. Eur. J. Cell Biol. 72, 113–121. PubMed

Bengough A. G., Bransby M. F., Hans J., McKenna S. J., Roberts T. J., Valentine T. A. (2006). Root responses to soil physical conditions; growth dynamics from field to cell. J. Exp. Bot. 57, 437–447. 10.1093/jxb/erj003 PubMed DOI

Benková E., Hejatko J. (2009). Hormone interactions at the root apical meristem. Plant Mol. Biol. 69, 383–396. 10.1007/s11103-008-9393-6 PubMed DOI

Bisgrove S. R., Hable W. E., Kropf D. L. (2004). +TIPs and microtubule regulation. The beginning of the plus end in plants. Plant Physiol. 136, 3855–3863. 10.1104/pp.104.051037 PubMed DOI PMC

Bisgrove S. R., Lee Y. R., Liu B., Peters N. T., Kropf D. L. (2008). The microtubule plus-end binding protein EB1 functions in root responses to touch and gravity signals in Arabidopsis. Plant Cell 20, 396–410. 10.1105/tpc.107.056846 PubMed DOI PMC

Brandizzi F., Irons S. L., Evans D. E. (2012). The plant nuclear envelope: new prospects for a poorly understood structure. New Phytol. 163, 227–246. 10.1111/j.1469-8137.2004.01118.x PubMed DOI

Carpenter A. E., Jones T. R., Lamprecht M. R., Clarke C., Kang I. H., Friman O., et al. . (2006). CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 7:R100. 10.1186/gb-2006-7-10-r100 PubMed DOI PMC

Chan J., Calder G. M., Doonan J. H., Lloyd C. W. (2003). EB1 reveals mobile microtubule nucleation sites in Arabidopsis. Nat. Cell Biol. 5, 967–971. 10.1038/ncb1057 PubMed DOI

Chytilova E., Macas J., Sliwinska E., Rafelski S. M., Lambert G. M., Galbraith D. W. (2000). Nuclear dynamics in Arabidopsis thaliana. Mol. Biol. Cell 11, 2733–2741. 10.1091/mbc.11.8.2733 PubMed DOI PMC

Clough S. J., Bent A. F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743. 10.1046/j.1365-313x.1998.00343.x PubMed DOI

Davis A. M., Hall A., Millar A. J., Darrah C., Davis S. J. (2009). Protocol: Streamlined sub-protocols for floral-dip transformation and selection of transformants in Arabidopsis thaliana. Plant Methods 5:3. 10.1186/1746-4811-5-3 PubMed DOI PMC

Dello Ioio R., Linhares F. S., Scacchi E., Casamitjana-Martinez E., Heidstra R., Costantino P., et al. . (2007). Cytokinins determine Arabidopsis root-meristem size by controlling cell differentiation. Curr. Biol. 17, 678–682. 10.1016/j.cub.2007.02.047 PubMed DOI

Dello Ioio R., Nakamura K., Moubayidin L., Perilli S., Taniguchi M., Morita M. T., et al. . (2008). A genetic framework for the control of cell division and differentiation in the root meristem. Science 322, 1380–1384. 10.1126/science.1164147 PubMed DOI

del Pozo J. C., Diaz-Trivino S., Cisneros N., Gutierrez C. (2006). The balance between cell division and endoreplication depends on E2FC-DPB, transcription factors regulated by the ubiquitin-SCFSKP2A pathway in Arabidopsis. Plant Cell 18, 2224–2235. 10.1105/tpc.105.039651 PubMed DOI PMC

Ding D., Muthuswamy S., Meier I. (2012). Functional interaction between the Arabidopsis orthologs of spindle assembly checkpoint proteins MAD1 and MAD2 and the nucleoporin NUA. Plant Mol. Biol. 79, 203–216 10.1007/s11103-012-9903-4 PubMed DOI

Dixit R., Chang E., Cyr R. (2006). Establishment of polarity during organization of the acentrosomal plant cortical microtubule array. Mol. Biol. Cell 17, 1298–1305. 10.1091/mbc.E05-09-0864 PubMed DOI PMC

Doskocilová A., Kohoutová L., Volc J., Kourová H., Benadá O., Chumová J., et al. . (2013). NITRILASE1 regulates the exit from proliferation, genome stability and plant development. New Phytol. 198, 685–698. 10.1111/nph.12185 PubMed DOI

Draviam V. M., Shapiro I., Aldridge B., Sorger P. K. (2006). Misorientation and reduced stretching of aligned sister kinetochores promote chromosome missegregation in EB1- or APC-depleted cells. EMBO J. 25, 2814–2827. 10.1038/sj.emboj.7601168 PubMed DOI PMC

Eleftheriou E. P., Adamakis I. D., Panteris E., Fatsiou M. (2015). Chromium-induced ultrastructural changes and oxidative stress in roots of Arabidopsis thaliana. Int. J. Mol. Sci. 16, 15852–15871. 10.3390/ijms160715852 PubMed DOI PMC

Hamada T. (2007). Microtubule-associated proteins in higher plants. J. Plant Res. 120, 79–98. 10.1007/s10265-006-0057-9 PubMed DOI

Hayashi K., Hasegawa J., Matsunaga S. (2013). The boundary of the meristematic and elongation zones in roots: endoreduplication precedes rapid cell expansion. Sci. Rep. 3, 2723. 10.1038/srep02723 PubMed DOI PMC

Heyman J., Van den Daele H., De Wit K., Boudolf V., Berckmans B., Verkest A., et al. . (2011). Arabidopsis ULTRAVIOLET-B-INSENSITIVE4 maintains cell division activity by temporal inhibition of the anaphase-promoting complex/cyclosome. Plant Cell 23, 4394–4410. 10.1105/tpc.111.091793 PubMed DOI PMC

Ho C. M., Hotta T., Guo F., Roberson R. W., Lee Y. R., Liu B. (2011). Interaction of antiparallel microtubules in the phragmoplast is mediated by the microtubule-associated protein MAP65-3 in Arabidopsis. Plant Cell 23, 2909–2923. 10.1105/tpc.110.078204 PubMed DOI PMC

Illés P., Schlicht M., Pavlovkin J., Lichtscheidl I., Baluska F., Ovecka M. (2006). Aluminium toxicity in plants: internalization of aluminium into cells of the transition zone in Arabidopsis root apices related to changes in plasma membrane potential, endosomal behaviour, and nitric oxide production. J. Exp. Bot. 57, 4201–4213. 10.1093/jxb/erl197 PubMed DOI

Ishida T., Adachi S., Yoshimura M., Shimizu K., Umeda M., Sugimoto K. (2010). Auxin modulates the transition from the mitotic cycle to the endocycle in Arabidopsis. Development 137, 63–71. 10.1242/dev.035840 PubMed DOI

Ishida T., Fujiwara S., Miura K., Stacey N., Yoshimura M., Schneider K., et al. . (2009). SUMO E3 ligase HIGH PLOIDY2 regulates endocycle onset and meristem maintenance in Arabidopsis. Plant Cell 21, 2284–2297. 10.1105/tpc.109.068072 PubMed DOI PMC

Ishikawa H., Evans M. L. (1993). The role of the distal elongation zone in the response of maize roots to auxin and gravity. Plant Physiol. 102, 1203–1210. PubMed PMC

Jiang K., Akhmanova A. (2011). Microtubule tip-interacting proteins: a view from both ends. Curr. Opin. Cell Biol. 23, 94–101. 10.1016/j.ceb.2010.08.008 PubMed DOI

Jin K., Shen J., Ashton R. W., White R. P., Dodd I. C., Phillips A. L., et al. . (2015). The effect of impedance to root growth on plant architecture in wheat. Plant Soil 392, 323–332. 10.1007/s11104-015-2462-0 PubMed DOI PMC

Ketelaar T., Faivre-Moskalenko C., Esseling J. J., de Ruijter N. C., Grierson C. S., Dogterom M., et al. . (2002). Positioning of nuclei in Arabidopsis root hairs: an actin-regulated process of tip growth. Plant Cell 14, 2941–2955. 10.1105/tpc.005892 PubMed DOI PMC

Kohoutová L., Kourová H., Nagy S. K., Volc J., Halada P., Mészáros T., et al. . (2015). The Arabidopsis mitogen-activated protein kinase 6 is associated with gamma-tubulin on microtubules, phosphorylates EB1c and maintains spindle orientation under nitrosative stress. New Phytol. 207, 1061–1074. 10.1111/nph.13501 PubMed DOI

Komaki S., Abe T., Coutuer S., Inzé D., Russinova E., Hashimoto T. (2010). Nuclear-localized subtype of end-binding 1 protein regulates spindle organization in Arabidopsis. J. Cell Sci. 123, 451–459. 10.1242/jcs.062703 PubMed DOI

Komarova Y., De Groot C. O., Grigoriev I., Gouveia S. M., Munteanu E. L., Schober J. M., et al. . (2009). Mammalian end binding proteins control persistent microtubule growth. J. Cell Biol. 184, 691–706. 10.1083/jcb.200807179 PubMed DOI PMC

la Cour T., Kiemer L., Molgaard A., Gupta R., Skriver K., Brunak S. (2004). Analysis and prediction of leucine-rich nuclear export signals. Protein Eng. Des. Sel. 17, 527–536. 10.1093/protein/gzh062 PubMed DOI

Lamprecht M. R., Sabatini D. M., Carpenter A. E. (2007). CellProfiler: free, versatile software for automated biological image analysis. Biotechniques 42, 71–75. 10.2144/000112257 PubMed DOI

Maizel A., von Wangenheim D., Federici F., Haseloff J., Stelzer E. H. (2011). High-resolution live imaging of plant growth in near physiological bright conditions using light sheet fluorescence microscopy. Plant J. 68, 377–385. 10.1111/j.1365-313X.2011.04692.x PubMed DOI

Mathur J., Mathur N., Kernebeck B., Srinivas B. P., Hulskamp M. (2003). A novel localization pattern for an EB1-like protein links microtubule dynamics to endomembrane organization. Curr. Biol. 13, 1991–1997. 10.1016/j.cub.2003.10.033 PubMed DOI

McLamore E. S., Diggs A., Calvo Marzal P., Shi J., Blakeslee J. J., Peer W. A., et al. . (2010). Non-invasive quantification of endogenous root auxin transport using an integrated flux microsensor technique. Plant J. 63, 1004–1016. 10.1111/j.1365-313X.2010.04300.x PubMed DOI

Okamoto T., Tsurumi S., Shibasaki K., Obana Y., Takaji H., Oono Y., et al. . (2008). Genetic dissection of hormonal responses in the roots of Arabidopsis grown under continuous mechanical impedance. Plant Physiol. 146, 1651–1662. 10.1104/pp.107.115519 PubMed DOI PMC

Ovecka M., Vaskebova L., Komis G., Luptovciak I., Smertenko A., Samaj J. (2015). Preparation of plants for developmental and cellular imaging by light-sheet microscopy. Nat. Protoc. 10, 1234–1247. 10.1038/nprot.2015.081 PubMed DOI

Paganelli L., Caillaud M. C., Quentin M., Damiani I., Govetto B., Lecomte P., et al. . (2015). Three BUB1 and BUBR1/MAD3-related spindle assembly checkpoint proteins are required for accurate mitosis in Arabidopsis. New Phytol. 205, 202–215 10.1111/nph.13073 PubMed DOI

Panteris E., Adamakis I. D., Daras G., Hatzopoulos P., Rigas S. (2013). Differential responsiveness of cortical microtubule orientation to suppression of cell expansion among the developmental zones of Arabidopsis thaliana root apex. PLoS ONE 8:e82442. 10.1371/journal.pone.0082442 PubMed DOI PMC

Petricka J. J., Schauer M. A., Megraw M., Breakfield N. W., Thompson J. W., Georgiev S., et al. . (2012). The protein expression landscape of the Arabidopsis root. Proc. Natl. Acad. Sci. U.S.A. 109, 6811–6818. 10.1073/pnas.1202546109 PubMed DOI PMC

Ren J., Longping W., Xinjiao G., Changjiang J., Yu X., Xuebiao Y. (2009). DOG1.0: illustrator of protein domain structures. Cell Res. 19, 271–273. 10.1038/cr.2009.6 PubMed DOI

Ruzicka K., Simaskova M., Duclercq J., Petrasek J., Zazimalova E., Simon S., et al. . (2009). Cytokinin regulates root meristem activity via modulation of the polar auxin transport. Proc. Natl. Acad. Sci. U.S.A. 106, 4284–4289. 10.1073/pnas.0900060106 PubMed DOI PMC

Samaj J., Baluska F., Voigt B., Schlicht M., Volkmann D., Menzel D. (2004). Endocytosis, actin cytoskeleton, and signaling. Plant Physiol. 135, 1150–1161. 10.1104/pp.104.040683 PubMed DOI PMC

Samajova O., Komis G., Samaj J. (2013). Emerging topics in the cell biology of mitogen-activated protein kinases. Trends Plant Sci. 18, 140–148. 10.1016/j.tplants.2012.11.004 PubMed DOI

Scheres B., Berleth T. (1998). Root development: new meanings for root canals? Curr. Opin. Plant Biol. 1, 32–36. 10.1016/S1369-5266(98)80124-6 PubMed DOI

Sena G., Frentz Z., Birnbaum K. D., Leibler S. (2011). Quantitation of cellular dynamics in growing Arabidopsis roots with light sheet microscopy. PLoS ONE 6:e21303. 10.1371/journal.pone.0021303 PubMed DOI PMC

Sliwinska E., Mathur J., Bewley J. D. (2012). Synchronously developing collet hairs in Arabidopsis thaliana provide an easily accessible system for studying nuclear movement and endoreduplication. J. Exp. Bot. 63, 4165–4178. 10.1093/jxb/ers099 PubMed DOI

Szklarczyk D., Franceschini A., Wyder S., Forslund K., Heller D., Huerta-Cepas J., et al. . (2015). STRING v10: protein-protein interaction networks, integrated over the tree of life. Nucleic Acids Res. 43, D447–D452. 10.1093/nar/gku1003 PubMed DOI PMC

Tamura N., Draviam V. M. (2012). Microtubule plus-ends within a mitotic cell are moving platforms' with anchoring, signalling and force-coupling roles. Open Biol. 2:120132. 10.1098/rsob.120132 PubMed DOI PMC

Tirnauer J. S., Canman J. C., Salmon E. D., Mitchison T. J. (2002a). EB1 targets to kinetochores with attached, polymerizing microtubules. Mol. Biol. Cell 13, 4308–4316. 10.1091/mbc.E02-04-0236 PubMed DOI PMC

Tirnauer J. S., Grego S., Salmon E. D., Mitchison T. J. (2002b). EB1-microtubule interactions in Xenopus egg extracts: role of EB1 in microtubule stabilization and mechanisms of targeting to microtubules. Mol. Biol. Cell 13, 3614–3626. 10.1091/mbc.02-04-0210 PubMed DOI PMC

van der Weele C. M., Jiang H. S., Palaniappan K. K., Ivanov V. B., Palaniappan K., Baskin T. I. (2003). A new algorithm for computational image analysis of deformable motion at high spatial and temporal resolution applied to root growth. Roughly uniform elongation in the meristem and also, after an abrupt acceleration, in the elongation zone. Plant Physiol. 132, 1138–1148. 10.1104/pp.103.021345 PubMed DOI PMC

Verbelen J. P., De Cnodder T., Le J., Vissenberg K., Baluska F. (2006). The root apex of Arabidopsis thaliana consists of four distinct zones of growth activities: meristematic zone, transition zone, fast elongation zone and growth terminating zone. Plant Signal. Behav. 1, 296–304. 10.4161/psb.1.6.3511 PubMed DOI PMC

Weigel D., Jurgens G. (2002). Stem cells that make stems. Nature 415, 751–754. 10.1038/415751a PubMed DOI

Analysis of formin functions during cytokinesis using specific inhibitor SMIFH2

Spatiotemporal Pattern of Ectopic Cell Divisions Contribute to Mis-Shaped Phenotype of Primary and Lateral Roots of katanin1 Mutant

Advances in Imaging Plant Cell Dynamics

Alfalfa Root Growth Rate Correlates with Progression of Microtubules during Mitosis and Cytokinesis as Revealed by Environmental Light-Sheet Microscopy