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• W3012381373 abstract "•An hyper-response to mechanical stress can lead to increased phenotypic variability•NEK6 exhibits a bipolar recruitment on microtubules that align with tensile stress•The nek6 wavy phenotype is consistent with a hyper-proprioceptive response•Growth rate uncouples shape- and growth-derived stress response in nek6 Growth variability generates mechanical conflicts in tissues. In plants, cortical microtubules usually align with maximal tensile stress direction, thereby mechanically reinforcing cell walls, and channeling growth rate and direction. How this is achieved remains largely unknown and likely involves microtubule regulators. The NIMA-related microtubule-associated kinase NEK6 phosphorylates tubulin, leading to the depolymerization of a subset of microtubules. We found that cortical microtubules exhibit a hyper-response to mechanical stress in the nek6 mutant. This response contributes to local cell protrusions in slow-growing regions of the nek6 mutant hypocotyl. When growth amplitude is higher, the hyper-alignment of microtubules leads to variable, stop-and-go, phenotypes, resulting in wavy hypocotyl shapes. After gravistimulation or touch, the nek6 mutant also exhibits a hyperbent hypocotyl phenotype, consistent with an enhanced perception of its own deformation. Strikingly, we find that NEK6 exhibits a novel form of polarity, being recruited at the ends of a subset of microtubules at cell edges. This pattern can be modified after local ablation, matching the new maximal tensile stress directions. We propose that NEK6 depolymerizes cortical microtubules that best align with maximal tensile stress to generate a noisier network of microtubules. This prevents an overreaction of microtubules to growth fluctuations and, instead, promotes the buffering of growth variations. Growth variability generates mechanical conflicts in tissues. In plants, cortical microtubules usually align with maximal tensile stress direction, thereby mechanically reinforcing cell walls, and channeling growth rate and direction. How this is achieved remains largely unknown and likely involves microtubule regulators. The NIMA-related microtubule-associated kinase NEK6 phosphorylates tubulin, leading to the depolymerization of a subset of microtubules. We found that cortical microtubules exhibit a hyper-response to mechanical stress in the nek6 mutant. This response contributes to local cell protrusions in slow-growing regions of the nek6 mutant hypocotyl. When growth amplitude is higher, the hyper-alignment of microtubules leads to variable, stop-and-go, phenotypes, resulting in wavy hypocotyl shapes. After gravistimulation or touch, the nek6 mutant also exhibits a hyperbent hypocotyl phenotype, consistent with an enhanced perception of its own deformation. Strikingly, we find that NEK6 exhibits a novel form of polarity, being recruited at the ends of a subset of microtubules at cell edges. This pattern can be modified after local ablation, matching the new maximal tensile stress directions. We propose that NEK6 depolymerizes cortical microtubules that best align with maximal tensile stress to generate a noisier network of microtubules. This prevents an overreaction of microtubules to growth fluctuations and, instead, promotes the buffering of growth variations. Robustness in living organisms emerges from coupling intrinsically suboptimal processes, be it at molecular scales, at the level of gene regulatory networks (e.g., [1Goentoro L. Shoval O. Kirschner M.W. Alon U. The incoherent feedforward loop can provide fold-change detection in gene regulation.Mol. Cell. 2009; 36: 894-899Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar]) or of cell/cell interactions. Mechanical forces have been proposed to add robustness to cell functions, notably by providing synchronizing cues for adjacent cells and constraining molecular outputs [2Davidson L.A. Mechanical design in embryos: mechanical signalling, robustness and developmental defects.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2017; 372: 20150516Crossref PubMed Scopus (24) Google Scholar, 3Hamant O. Moulia B. How do plants read their own shapes?.New Phytol. 2016; 212: 333-337Crossref PubMed Scopus (47) Google Scholar]. Because plant cells do not migrate, final organ shape is a good proxy for robustness at multiple scales, from molecules to multicellular forms. More specifically, the mechanics behind cell shapes mainly rely on cell walls, and, within the cell wall, cellulose microfibrils appear as the main load-bearing components [4Cosgrove D.J. Growth of the plant cell wall.Nat. Rev. Mol. Cell Biol. 2005; 6: 850-861Crossref PubMed Scopus (2275) Google Scholar]. Because cortical microtubules (CMTs) usually guide the deposition of cellulose in cell walls, CMT behavior is a good proxy for final plant cell and tissue shapes. Last, cell walls are constantly under high tensile stress, being pulled by turgor pressure in the MPa range. Interestingly, CMTs have been shown to align with maximal tensile stress direction in most aerial plant organs [5Green P. King A. A mechanism for the origin of specifically oriented textures in development with special reference to Nitella wall texture.Aust. J. Biol. Sci. 1966; 19: 421-437Crossref Google Scholar, 6Hamant O. Heisler M.G. Jönsson H. Krupinski P. Uyttewaal M. Bokov P. Corson F. Sahlin P. Boudaoud A. Meyerowitz E.M. et al.Developmental patterning by mechanical signals in Arabidopsis.Science. 2008; 322: 1650-1655Crossref PubMed Scopus (648) Google Scholar, 7Sampathkumar A. Krupinski P. Wightman R. Milani P. Berquand A. Boudaoud A. Hamant O. Jönsson H. Meyerowitz E.M. Subcellular and supracellular mechanical stress prescribes cytoskeleton behavior in Arabidopsis cotyledon pavement cells.eLife. 2014; 3: e01967Crossref PubMed Google Scholar, 8Robinson S. Kuhlemeier C. Global compression reorients cortical microtubules in Arabidopsis hypocotyl epidermis and promotes growth.Curr. Biol. 2018; 28: 1794-1802Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 9Verger S. Long Y. Boudaoud A. Hamant O. A tension-adhesion feedback loop in plant epidermis.eLife. 2018; 7: e34460Crossref PubMed Scopus (64) Google Scholar]. This response has been involved in the maintenance of cell shapes [7Sampathkumar A. Krupinski P. Wightman R. Milani P. Berquand A. Boudaoud A. Hamant O. Jönsson H. Meyerowitz E.M. Subcellular and supracellular mechanical stress prescribes cytoskeleton behavior in Arabidopsis cotyledon pavement cells.eLife. 2014; 3: e01967Crossref PubMed Google Scholar, 10Sapala A. Runions A. Routier-Kierzkowska A.-L. Das Gupta M. Hong L. Hofhuis H. et al.Why plants make puzzle cells, and how their shape emerges.eLife. 2018; 7: e32794Crossref PubMed Scopus (122) Google Scholar], in monitoring growth heterogeneity [11Uyttewaal M. Burian A. Alim K. Landrein B. Borowska-Wykręt D. Dedieu A. Peaucelle A. Ludynia M. Traas J. Boudaoud A. et al.Mechanical stress acts via katanin to amplify differences in growth rate between adjacent cells in Arabidopsis.Cell. 2012; 149: 439-451Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 12Hervieux N. Tsugawa S. Fruleux A. Dumond M. Routier-Kierzkowska A.-L. Komatsuzaki T. et al.Mechanical shielding of rapidly growing cells buffers growth heterogeneity and contributes to organ shape reproducibility.Curr. Biol. 2017; 27: 3468-3479Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar] and in restricting organ growth [13Hervieux N. Dumond M. Sapala A. Routier-Kierzkowska A.-L. Kierzkowski D. Roeder A.H.K. et al.A mechanical feedback restricts sepal growth and shape in Arabidopsis.Curr. Biol. 2016; 26: 1019-1028Abstract Full Text Full Text PDF Scopus (134) Google Scholar]. The CMT-plant shape nexus is thus key to morphogenesis and developmental robustness. Here, we observed that mutation in a microtubule kinase confers hyper-alignment of microtubules, and we relate this behavior to mechanical stress and growth. Many Arabidopsis mutants impaired in microtubule dynamics exhibit shape defects. This is notably the case of the katanin mutant, which exhibits reduced microtubule self-organization abilities, due to reduced severing frequency, and thus a slower response of microtubules to mechanical stress [11Uyttewaal M. Burian A. Alim K. Landrein B. Borowska-Wykręt D. Dedieu A. Peaucelle A. Ludynia M. Traas J. Boudaoud A. et al.Mechanical stress acts via katanin to amplify differences in growth rate between adjacent cells in Arabidopsis.Cell. 2012; 149: 439-451Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 14Bichet A. Desnos T. Turner S. Grandjean O. Höfte H. BOTERO1 is required for normal orientation of cortical microtubules and anisotropic cell expansion in Arabidopsis.Plant J. 2001; 25: 137-148Crossref PubMed Google Scholar]. These defects result in the formation of isotropic CMT arrays in most cells, isotropic growth, and impaired organ elongation. The Arabidopsis NIMA (Never In Mitosis A)-related kinase 6 (NEK6) is also involved in directional cell elongation through its impact on microtubule organization [15Motose H. Tominaga R. Wada T. Sugiyama M. Watanabe Y. A NIMA-related protein kinase suppresses ectopic outgrowth of epidermal cells through its kinase activity and the association with microtubules.Plant J. 2008; 54: 829-844Crossref PubMed Scopus (34) Google Scholar, 16Motose H. Hamada T. Yoshimoto K. Murata T. Hasebe M. Watanabe Y. et al.NIMA-related kinases 6, 4, and 5 interact with each other to regulate microtubule organization during epidermal cell expansion in Arabidopsis thaliana.Plant J. 2011; 67: 993-1005Crossref PubMed Scopus (39) Google Scholar, 17Takatani S. Ozawa S. Yagi N. Hotta T. Hashimoto T. Takahashi Y. Takahashi T. Motose H. Directional cell expansion requires NIMA-related kinase 6 (NEK6)-mediated cortical microtubule destabilization.Sci. Rep. 2017; 7: 7826Crossref PubMed Scopus (11) Google Scholar, 18Sakai T. Honing Hv. Nishioka M. Uehara Y. Takahashi M. Fujisawa N. Saji K. Seki M. Shinozaki K. Jones M.A. et al.Armadillo repeat-containing kinesins and a NIMA-related kinase are required for epidermal-cell morphogenesis in Arabidopsis.Plant J. 2008; 53: 157-171Crossref PubMed Scopus (79) Google Scholar]. NEK6 kinase activity and localization on microtubules are essential for cell elongation [15Motose H. Tominaga R. Wada T. Sugiyama M. Watanabe Y. A NIMA-related protein kinase suppresses ectopic outgrowth of epidermal cells through its kinase activity and the association with microtubules.Plant J. 2008; 54: 829-844Crossref PubMed Scopus (34) Google Scholar]. CMTs in nek6 mutant are resistant to microtubule depolymerization drugs, suggesting that NEK6 destabilizes CMTs [16Motose H. Hamada T. Yoshimoto K. Murata T. Hasebe M. Watanabe Y. et al.NIMA-related kinases 6, 4, and 5 interact with each other to regulate microtubule organization during epidermal cell expansion in Arabidopsis thaliana.Plant J. 2011; 67: 993-1005Crossref PubMed Scopus (39) Google Scholar]. NEK6-mediated β-tubulin phosphorylation prevents polymerization of aberrant microtubules, suggesting that NEK6 depolymerizes specific types of CMTs [17Takatani S. Ozawa S. Yagi N. Hotta T. Hashimoto T. Takahashi Y. Takahashi T. Motose H. Directional cell expansion requires NIMA-related kinase 6 (NEK6)-mediated cortical microtubule destabilization.Sci. Rep. 2017; 7: 7826Crossref PubMed Scopus (11) Google Scholar]. A recent study of the basal land plant Marchantia polymorpha further demonstrates the evolutionarily conserved NEK-dependent mechanism of directional cell growth through microtubule depolymerization [19Otani K. Ishizaki K. Nishihama R. Takatani S. Kohchi T. Takahashi T. et al.An evolutionarily conserved NIMA-related kinase directs rhizoid tip growth in the basal land plant Marchantia polymorpha.Development. 2018; 145: 154617Crossref Scopus (16) Google Scholar]. The biochemical characterization of NEK6 thus offers the unique opportunity to investigate the contribution of microtubule polymerization rate in the microtubule response to stress. The possibility to modify microtubule dynamics in a novel way could also help us identify new functions, and possibly an optimum, for the microtubule response to stress. Here, through mechanical perturbation and live imaging, we show that NEK6 reduces the microtubule response to stress; this also reduces proprioception, thereby smoothing tissue and cell-growth patterns in the hypocotyl. Previously, we reported that NEK6 is required for both directional cell growth and organ development ([20Motose H. Takatani S. Ikeda T. Takahashi T. NIMA-related kinases regulate directional cell growth and organ development through microtubule function in Arabidopsis thaliana.Plant Signal. Behav. 2012; 7: 1552-1555Crossref PubMed Scopus (16) Google Scholar], Figures 1A and 1B ; Figures S1F and S1G). To investigate precisely the organ-scale defect of nek6-1 mutant, seedling growth was recorded over time (Figures 1A and 1B; Video S1). The wild type exhibited slight fluctuations in its growth trajectory; in contrast, the nek6-1 mutant displayed marked growth deviations: the nek6-1 hypocotyls did not grow straight (Figure S1B, NWT = 74, Nnek6 = 80, p = 4.0 × 10−9 t test). This waving phenotype was mostly restricted to the rootward region of the hypocotyl, i.e., in the bottom half of the hypocotyl ([9Verger S. Long Y. Boudaoud A. Hamant O. A tension-adhesion feedback loop in plant epidermis.eLife. 2018; 7: e34460Crossref PubMed Scopus (64) Google Scholar], Figures 1A and 1B). As reported before, we could also observe ectopic outgrowth of epidermal cells [15Motose H. Tominaga R. Wada T. Sugiyama M. Watanabe Y. A NIMA-related protein kinase suppresses ectopic outgrowth of epidermal cells through its kinase activity and the association with microtubules.Plant J. 2008; 54: 829-844Crossref PubMed Scopus (34) Google Scholar, 18Sakai T. Honing Hv. Nishioka M. Uehara Y. Takahashi M. Fujisawa N. Saji K. Seki M. Shinozaki K. Jones M.A. et al.Armadillo repeat-containing kinesins and a NIMA-related kinase are required for epidermal-cell morphogenesis in Arabidopsis.Plant J. 2008; 53: 157-171Crossref PubMed Scopus (79) Google Scholar]. Interestingly, these were instead mainly located in the shootward region of the hypocotyl (Figure S1A). eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI0NjE4NDlmYzA3OWU0YmNlOWI0MGMwZGViYjlhY2M4NSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc5MDMzNDYzfQ.rNxw_DQbWaUIhabN7pZEX_IoHAhSaWBdN7gtvmCRViYGPtseh3V6RdnUZebVqSeEkBHDFAL2rGq9jQDM_Dk8pvG88y6r-kqBrDz8PV3FdOAmLKDwuA8mEJTdES7GF1mM_joRoeJ7Gsh78Jehh4sZCVf8lBWhCKENJCaP4sDTGoGxqwSFumfBgEZj0vfXRPCYM518x9LeRUDflNG3hw5dyd9dL5SEP57RYrz-wjlOjgUItV6jx24u_0YGr2CPtly0us2b56O19Fwhf948dM_CSUNHa9fdlE0QfGiMMRITEMdkEyyrAPQaElVPnKMP3hvNO3_yPZDJ8cTQyMfZh8uPRQ Download .mp4 (1.01 MB) Help with .mp4 files Video S1. Time-Lapse Video of 2- to 3-Day-Old Wild-Type and nek6-1 Seedlings, Related to Figures 1 and S1Wider view of time-lapse observation shown in Figure 1A. Scale bar, 1 mm To quantify this phenotype, we defined waviness as the ratio between hypocotyl length and the shortest, straight, distance between seed and cotyledon (Figure 1C). The hypocotyl exhibits a well-established and stereotypical growth gradient, where rootward cells are growing much faster than shootward cells [9Verger S. Long Y. Boudaoud A. Hamant O. A tension-adhesion feedback loop in plant epidermis.eLife. 2018; 7: e34460Crossref PubMed Scopus (64) Google Scholar, 21Gendreau E. Traas J. Desnos T. Grandjean O. Caboche M. Höfte H. Cellular basis of hypocotyl growth in Arabidopsis thaliana.Plant Physiol. 1997; 114: 295-305Crossref PubMed Scopus (505) Google Scholar]. Differences in growth defects along the nek6 hypocotyl may be due to such prepattern. To test that hypothesis, we first correlated hypocotyl length and waviness in nek6. 7-day-old seedlings differ in hypocotyl length even within the same line and such natural variation is large enough to draw a correlation. We found that shorter hypocotyls were less wavy. On the contrary, longer hypocotyls were wavier (Figure 1C; Pearson’s correlation, nWT = 53 r = 0.34, p = 0.013, nnek6-1 = 64, r = 0.55, p = 2.5 × 10−6). As cell divisions are rare in hypocotyls [21Gendreau E. Traas J. Desnos T. Grandjean O. Caboche M. Höfte H. Cellular basis of hypocotyl growth in Arabidopsis thaliana.Plant Physiol. 1997; 114: 295-305Crossref PubMed Scopus (505) Google Scholar], this shows that, in nek6, wavy growth correlates with rapid cell elongation. Next, we counted the number of ectopic outgrowth in the shootward (top half) and rootward (bottom half) regions of the hypocotyl using a previously reported method [15Motose H. Tominaga R. Wada T. Sugiyama M. Watanabe Y. A NIMA-related protein kinase suppresses ectopic outgrowth of epidermal cells through its kinase activity and the association with microtubules.Plant J. 2008; 54: 829-844Crossref PubMed Scopus (34) Google Scholar]. This analysis confirmed that the number of ectopic outgrowth is increased in the shootward region of the hypocotyl, where cell growth is reported slower (Figure S1C). In contrast to hypocotyl waviness, the presence of ectopic outgrowths correlates with slower growth. To further test this correlation, we stimulated hypocotyl elongation by growing seedlings in the dark. In these conditions, wild-type and nek6-1 seedlings exhibited growth deviations throughout the length of their hypocotyls (Figures S1D and S1E). Interestingly, ectopic outgrowth was completely suppressed in dark-grown nek6-1 mutant (Figure S1E). This further confirms that ectopic outgrowth and hypocotyl waviness correlate with slow growth and fast growth, respectively. Next, we tested whether the nek6 mutation induces or amplifies a pre-existing wavy growth pattern. To do so, we imposed external constraints on hypocotyl growth. First, we gravistimulated young nek6-1 seedlings. We reasoned that if the nek6 mutation induces wavy growth independent of the hypocotyl’s normal growth pattern, the response of the hypocotyl to gravity in nek6 should be noisier, whereas, if the nek6 mutation amplifies the wild-type response to gravity, the nek6 hypocotyls should be more curved than the wild type. Seedlings were grown on agar medium in the dark and rotated by 90 degrees from the original orientation to induce gravistimulation (Figure 1D). After gravistimulation, seedlings grew upward against gravity in both the wild-type and nek6-1 mutant 3 h after gravistimulation. However, nek6-1 mutant seedlings displayed more curvy hypocotyls 16 h after gravistimulation, when compared to the wild type (Figure 1D). The difference between wild-type and mutant waviness was significant (Figure 1H, nWT = 12, nnek6-1 = 13, p = 0.026 Mann-Whitney U test). Some nek6-1 mutant hypocotyls were even curved at an angle superior to 90 degrees (Figure 1E). Note that the nek6-1 mutant could respond to gravity as quickly as the wild type (Figure S1H, nWT = 10, nnek6-1 = 7, n.s. p > 0.05 t test). Thus the nek6-1 mutant is not agravitropic, but it exhibits a specific defect in growth direction rather than gravitropism. To further confirm that the nek6-1 mutant amplifies wild-type growth patterns, we exposed nek6-1 seedling to a touch stimulus. When slanting plates, roots touch the medium as they grow, and this modifies their trajectory (e.g., [22Okada K. Shimura Y. Reversible root tip rotation in Arabidopsis seedlings induced by obstacle-touching stimulus.Science. 1990; 250: 274-276Crossref PubMed Scopus (248) Google Scholar]). We performed the same test for dark-grown hypocotyls. Agar medium containing plates were inclined forward with an angle of 30 degrees to induce upward bending growth of hypocotyls and continuous mechanical friction between seedlings and agar medium (Figures 1F and 1G). In these conditions, wild-type hypocotyl growth was slightly skewed, whereas nek6-1 hypocotyls were very curvy, sometimes even exhibiting a full loop phenotype (Figure 1G). Wild-type and mutant exhibited significant difference in their waviness (Figure 1I; vertical, nWT = 19, nnek6-1 = 19 p = 5.7 × 10−11; oblique, nWT = 22, nnek6-1 = 23, p = 7.1 × 10−6 Mann-Whitney U test). Altogether, these observations support a scenario in which growth deviations are amplified in the nek6 mutant. Because of the biochemical function of NEK6 as a tubulin kinase and knowing that CMTs are major regulators of growth direction, we next checked the microtubule organization in the basal region of nek6 hypocotyls. In the wild type, CMTs display rotating or unstable organizations in the fast-growing region of the hypocotyl [23Chan J. Calder G. Fox S. Lloyd C. Cortical microtubule arrays undergo rotary movements in Arabidopsis hypocotyl epidermal cells.Nat. Cell Biol. 2007; 9: 171-175Crossref PubMed Scopus (116) Google Scholar, 24Vineyard L. Elliott A. Dhingra S. Lucas J.R. Shaw S.L. Progressive transverse microtubule array organization in hormone-induced Arabidopsis hypocotyl cells.Plant Cell. 2013; 25: 662-676Crossref PubMed Scopus (54) Google Scholar], and they become predominantly longitudinal later on as growth slows down [25Baskin T.I. Anisotropic expansion of the plant cell wall.Annu. Rev. Cell Dev. Biol. 2005; 21: 203-222Crossref PubMed Scopus (410) Google Scholar, 26Crowell E.F. Timpano H. Desprez T. Franssen-Verheijen T. Emons A.-M. Höfte H. Vernhettes S. Differential regulation of cellulose orientation at the inner and outer face of epidermal cells in the Arabidopsis hypocotyl.Plant Cell. 2011; 23: 2592-2605Crossref PubMed Scopus (97) Google Scholar, 27Chan J. Eder M. Crowell E.F. Hampson J. Calder G. Lloyd C. Microtubules and CESA tracks at the inner epidermal wall align independently of those on the outer wall of light-grown Arabidopsis hypocotyls.J. Cell Sci. 2011; 124: 1088-1094Crossref PubMed Scopus (58) Google Scholar]. To quantify CMT behavior in hypocotyl cells, we used the ImageJ macro FibrilTool, which is based on the concept of nematic tensors and measures the average orientation and anisotropy of CMT arrays [28Boudaoud A. Burian A. Borowska-Wykręt 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: 457-463Crossref PubMed Scopus (342) Google Scholar]. All numbers in this article relate to microtubule orientation and anisotropy per cell. The nek6-1 mutant exhibited more consistent CMT orientations than the wild type, especially in the rootward part of the hypocotyl (Figure S2). Overall, the standard deviation of CMT orientation was smaller in nek6-1 than in the wild type (Figure S2E, nWT = 13, nnek6-1 = 11, p = 0.033 t test). Observation of the same cells over 2 h confirmed that CMTs in the wild type continuously change their orientation (Figure 2A). Although we could detect a bias toward the longitudinal axis when averaging CMT orientations, microtubule arrays in the wild type could completely change their orientation in only 1 h (Figure 2B). In contrast, most CMTs aligned longitudinally in the nek6-1 mutant (Figure 2B; Figure S2), and CMT reorientations were dramatically reduced (Figure 2C). When pooling the quantifications together, we found that the reorientation angle of CMTs in 1 h was significantly smaller in nek6 than in the wild type (Figure 2D, nWT = 136, nnek6-1 = 128 p = 1.5 × 10−8 Mann-Whitney U test). Therefore, the nek6 mutation seems to reduce the deviations in CMT behavior in the fast-growing region of the hypocotyl. Altogether, our data suggest that, in the nek6 mutant, the CMT pattern is denoised, and this amplifies the hypocotyl response to growth fluctuations. The presence of longitudinal microtubules after a fast-growing phase in hypocotyls has been related to the pattern of tensile stress in the hypocotyl epidermis: the inner tissues would pull the epidermis along the axis of the hypocotyl, and the epidermis would resist by aligning CMTs (and reinforcing its outer walls) in the longitudinal direction [8Robinson S. Kuhlemeier C. Global compression reorients cortical microtubules in Arabidopsis hypocotyl epidermis and promotes growth.Curr. Biol. 2018; 28: 1794-1802Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar]. Such a stress pattern and CMT alignment have been further validated using mutants with cell-cell adhesion defects [9Verger S. Long Y. Boudaoud A. Hamant O. A tension-adhesion feedback loop in plant epidermis.eLife. 2018; 7: e34460Crossref PubMed Scopus (64) Google Scholar]. Given the hyper-alignment of longitudinal CMTs, and the overall hyper-response to growth fluctuations in nek6 (Figure S2), we next investigated whether CMTs in nek6 could be more sensitive to tensile stress than the wild type. To check this, we locally changed the stress pattern in hypocotyls by performing cell ablations. Because the epidermis is under tension, the pattern of stress becomes circumferential around the ablation site, and CMT orientation follow this pattern within 2 h [6Hamant O. Heisler M.G. Jönsson H. Krupinski P. Uyttewaal M. Bokov P. Corson F. Sahlin P. Boudaoud A. Meyerowitz E.M. et al.Developmental patterning by mechanical signals in Arabidopsis.Science. 2008; 322: 1650-1655Crossref PubMed Scopus (648) Google Scholar, 7Sampathkumar A. Krupinski P. Wightman R. Milani P. Berquand A. Boudaoud A. Hamant O. Jönsson H. Meyerowitz E.M. Subcellular and supracellular mechanical stress prescribes cytoskeleton behavior in Arabidopsis cotyledon pavement cells.eLife. 2014; 3: e01967Crossref PubMed Google Scholar, 8Robinson S. Kuhlemeier C. Global compression reorients cortical microtubules in Arabidopsis hypocotyl epidermis and promotes growth.Curr. Biol. 2018; 28: 1794-1802Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 9Verger S. Long Y. Boudaoud A. Hamant O. A tension-adhesion feedback loop in plant epidermis.eLife. 2018; 7: e34460Crossref PubMed Scopus (64) Google Scholar]. We confirmed this response in the wild type and observed that the response was more pronounced in the nek6-1 mutant, at least qualitatively (Figure 3A). We then quantified the anisotropy and average angle of the CMT arrays using FibrilTool, using the method described in [9Verger S. Long Y. Boudaoud A. Hamant O. A tension-adhesion feedback loop in plant epidermis.eLife. 2018; 7: e34460Crossref PubMed Scopus (64) Google Scholar] (Figures 3B and 3D; [28Boudaoud A. Burian A. Borowska-Wykręt 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: 457-463Crossref PubMed Scopus (342) Google Scholar]). We found that CMT array anisotropy is significantly increased in the nek6-1 mutant (Figure 3B, nWT = 164, nnek6-1 = 93, p0.5h = 4.7 × 10−4, p2.5h = 1.4 × 10−7 t test) and that CMT arrays around the ablation are significantly more circumferential in the mutant than in the wild type (Figure 3D nWT = 164, nnek6-1 = 93, p = 0.049 Kolmogorov-Smirnov test). Four hours after ablation, wild-type cells surrounding the ablation site already elongated toward the ablation site, matching the CMT orientation and acting as a wound healing mechanism (Figure S3). The reduction in hole area following wound closure was significantly different between WT and nek6-1 (Figure S3B, nWT = 10, nnek6-1 = 17, p2.5h = 0.042, p4.5h = 0.0024). Here again, cell elongation in nek6-1 was more pronounced, and wound healing was complete earlier than in the wild type (Figure S3). Note that, in nek6, the rapid reorientation of CMTs after ablation may appear contradictory with the stable CMT orientation in the hypocotyl (Figure 1). This is in fact consistent with the idea that CMT response to stress is more pronounced in nek6: in the hypocotyl, epidermal cells are continuously pulled by inner cells, whereas, after ablation, the stress pattern changes very rapidly. In other words, both sets of observations independently reflect a similar enhanced CMT response to stress in nek6. Altogether, these results further confirm that growth responses are amplified in nek6 and suggest that this behavior is linked to a hyper-response of microtubules to mechanical stress in nek6. If our hypothesis is true, a hyper-response to mechanical stress should also be consistent with the presence of ectopic outgrowth in the slow-growing shootward region of the hypocotyl in nek6. In contrast to the fast-growing region of the hypocotyl, and, as previously reported [17Takatani S. Ozawa S. Yagi N. Hotta T. Hashimoto T. Takahashi Y. Takahashi T. Motose H. Directional cell expansion requires NIMA-related kinase 6 (NEK6)-mediated cortical microtubule destabilization.Sci. Rep. 2017; 7: 7826Crossref PubMed Scopus (11) Google Scholar], CMTs appeared quite disorganized in the slow-growing region of the hypocotyl, both in the wild type and in nek6 (Figure 4A). However, we could detect a bias toward subcellular circumferential CMT organization in nek6-1 cells (Figure 4A). Circumferential CMTs were often observed in cells from the slow-growing shootward region where cell curvature is also stronger (Figure 4B, nWT = 178, nnek6-1 = 165). To quantify this phenotype, we set the boundary at cell position #8 (see Figure S2A) and counted the number of cells with circumferential CMTs in nek6. In the rootward region (cell position #1 to #8), 9 out of 80 cells exhibited circumferential CMTs. In contrast, in the shootward region (cell position #9 to #20), 23 out of 85 cells exhibited circumferential CMTs (Figure S4E, nWT = 178, nnek6-1 = 165). Assuming that the outer wall is under tension, in the most parsimonious scenario, the convex curvature of the wall would prescribe a circumferential stress pattern in" @default.
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• W3012381373 creator A5086744596 @default.
• W3012381373 date "2020-04-01" @default.
• W3012381373 modified "2023-09-30" @default.
• W3012381373 title "Microtubule Response to Tensile Stress Is Curbed by NEK6 to Buffer Growth Variation in the Arabidopsis Hypocotyl" @default.
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• W3012381373 doi "https://doi.org/10.1016/j.cub.2020.02.024" @default.
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