Phytohormone cytokinin guides microtubule dynamics during cell progression from proliferative to differentiated stage
2020; Springer Nature; Volume: 39; Issue: 17 Linguagem: Inglês
10.15252/embj.2019104238
ISSN1460-2075
AutoresJuan Carlos Montesinos, Anas Abuzeineh, Aglaja Kopf, Alba Juanes-García, Krisztina Ötvös, Jan Petrášek, Michael Sixt, Eva Benková,
Tópico(s)Plant nutrient uptake and metabolism
ResumoArticle15 July 2020Open Access Transparent process Phytohormone cytokinin guides microtubule dynamics during cell progression from proliferative to differentiated stage Juan Carlos Montesinos orcid.org/0000-0001-9179-6099 Institute of Science and Technology Austria (IST Austria), Klosterneuburg, Austria Search for more papers by this author Anas Abuzeineh orcid.org/0000-0001-5220-1005 Department of Plant Biotechnology and Bioinformatics, Ghent University and Center for Plant Systems Biology, VIB, Gent, Belgium Search for more papers by this author Aglaja Kopf orcid.org/0000-0002-2187-6656 Institute of Science and Technology Austria (IST Austria), Klosterneuburg, Austria Search for more papers by this author Alba Juanes-Garcia orcid.org/0000-0002-1009-9652 Institute of Science and Technology Austria (IST Austria), Klosterneuburg, Austria Search for more papers by this author Krisztina Ötvös orcid.org/0000-0002-5503-4983 Institute of Science and Technology Austria (IST Austria), Klosterneuburg, Austria Bioresources Unit, Center for Health & Bioresources, AIT Austrian Institute of Technology GmbH, Tulln, Austria Search for more papers by this author Jan Petrášek orcid.org/0000-0002-6719-2735 Institute of Experimental Botany, The Czech Academy of Sciences, Praha, Czech Republic Search for more papers by this author Michael Sixt orcid.org/0000-0002-6620-9179 Institute of Science and Technology Austria (IST Austria), Klosterneuburg, Austria Search for more papers by this author Eva Benková Corresponding Author [email protected] orcid.org/0000-0002-8510-9739 Institute of Science and Technology Austria (IST Austria), Klosterneuburg, Austria Search for more papers by this author Juan Carlos Montesinos orcid.org/0000-0001-9179-6099 Institute of Science and Technology Austria (IST Austria), Klosterneuburg, Austria Search for more papers by this author Anas Abuzeineh orcid.org/0000-0001-5220-1005 Department of Plant Biotechnology and Bioinformatics, Ghent University and Center for Plant Systems Biology, VIB, Gent, Belgium Search for more papers by this author Aglaja Kopf orcid.org/0000-0002-2187-6656 Institute of Science and Technology Austria (IST Austria), Klosterneuburg, Austria Search for more papers by this author Alba Juanes-Garcia orcid.org/0000-0002-1009-9652 Institute of Science and Technology Austria (IST Austria), Klosterneuburg, Austria Search for more papers by this author Krisztina Ötvös orcid.org/0000-0002-5503-4983 Institute of Science and Technology Austria (IST Austria), Klosterneuburg, Austria Bioresources Unit, Center for Health & Bioresources, AIT Austrian Institute of Technology GmbH, Tulln, Austria Search for more papers by this author Jan Petrášek orcid.org/0000-0002-6719-2735 Institute of Experimental Botany, The Czech Academy of Sciences, Praha, Czech Republic Search for more papers by this author Michael Sixt orcid.org/0000-0002-6620-9179 Institute of Science and Technology Austria (IST Austria), Klosterneuburg, Austria Search for more papers by this author Eva Benková Corresponding Author [email protected] orcid.org/0000-0002-8510-9739 Institute of Science and Technology Austria (IST Austria), Klosterneuburg, Austria Search for more papers by this author Author Information Juan Carlos Montesinos1, Anas Abuzeineh2, Aglaja Kopf1, Alba Juanes-Garcia1, Krisztina Ötvös1,3, Jan Petrášek4, Michael Sixt1 and Eva Benková *,1 1Institute of Science and Technology Austria (IST Austria), Klosterneuburg, Austria 2Department of Plant Biotechnology and Bioinformatics, Ghent University and Center for Plant Systems Biology, VIB, Gent, Belgium 3Bioresources Unit, Center for Health & Bioresources, AIT Austrian Institute of Technology GmbH, Tulln, Austria 4Institute of Experimental Botany, The Czech Academy of Sciences, Praha, Czech Republic *Corresponding author. Tel: +43 2243 9000 5301; E-mail: [email protected] EMBO J (2020)39:e104238https://doi.org/10.15252/embj.2019104238 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Cell production and differentiation for the acquisition of specific functions are key features of living systems. The dynamic network of cellular microtubules provides the necessary platform to accommodate processes associated with the transition of cells through the individual phases of cytogenesis. Here, we show that the plant hormone cytokinin fine-tunes the activity of the microtubular cytoskeleton during cell differentiation and counteracts microtubular rearrangements driven by the hormone auxin. The endogenous upward gradient of cytokinin activity along the longitudinal growth axis in Arabidopsis thaliana roots correlates with robust rearrangements of the microtubule cytoskeleton in epidermal cells progressing from the proliferative to the differentiation stage. Controlled increases in cytokinin activity result in premature re-organization of the microtubule network from transversal to an oblique disposition in cells prior to their differentiation, whereas attenuated hormone perception delays cytoskeleton conversion into a configuration typical for differentiated cells. Intriguingly, cytokinin can interfere with microtubules also in animal cells, such as leukocytes, suggesting that a cytokinin-sensitive control pathway for the microtubular cytoskeleton may be at least partially conserved between plant and animal cells. Synopsis Phytohormones auxin and cytokinin play antagonistic roles in plant development, including root growth. Here, cytokinin is found to fine-tune microtubule dynamics in Arabidopsis roots, as well as in animal cells. Cytokinin activity gradients in Arabidopsis thaliana roots correlate with gradual microtubule cytoskeleton reconfiguration along the longitudinal growth axis. Cytokinin promotes re-orientation of microtubules and reduces microtubule growth rate in root epidermal cells progressing from the proliferative-to-differentiation stage. Cytokinin signalling pathway components contribute to microtubule cytoskeleton regulation. Cytokinin counteracts auxin-driven microtubule rearrangements. Cytokinin can suppress microtubule growth in mammalian leukocytes. Introduction Growth and development of living organisms depend on the constant production of new cells that subsequently differentiate, thereby acquiring specific shapes and functions. The cytoskeleton provides an elementary framework for cell functions, including cell division, cell motility, cell shape, intracellular organization, and trafficking of organelles (Brandizzi & Wasteneys, 2013; Akhmanova & Steinmetz, 2015). Microtubules (MTs) as a major component of the eukaryotic cytoskeleton play important roles in virtually every aspect of its functions. To fulfill these diverse activities, MTs assemble into distinct arrays that are characterized by high dynamics (Horio & Murata, 2014). In plants, specialized tissues, called meristems, maintain the proliferative capacity and constantly produce new cells, thereby undergoing a gradual transformation from a proliferative to a fully differentiated stage. Typically, the progress of cells through the individual phases is spatio-temporally tightly controlled, resulting in the formation of discrete domains encompassing proliferation, transition, expansion/elongation, and differentiation in plant organs (Le et al, 2004; Hayashi et al, 2013). As cells progress from the proliferative to the differentiation stage, the microtubular cytoskeleton goes through substantial rearrangements to accommodate cyto-physiological changes that occur during cytogenesis. In dividing cells, MTs are involved in the formation of the preprophase band (PPB) in the cell equator, which predicts the future orientation of the division plane (de Keijzer et al, 2014; Hashimoto, 2015). MTs, as a component of the mitotic spindle, contribute to the chromosome separation and they participate in the formation of the cell plate that will separate two daughter cells (Hamada, 2014; Smertenko et al, 2017). In non-dividing cells, MTs are localized in the cell cortex, designated cortical microtubules (CMTs), and form arrays that are laterally anchored to the plasma membrane (Lucas & Shaw, 2008; Oda, 2015). As plant cells expand, differentiate, and acquire specific shapes, CMTs have an important function in the delivery and deposition of new cell wall components and in the cell shape maintenance (Hashimoto, 2015; Elliott & Shaw, 2018). Mutants in the core subunits of MTs or factors regulating the dynamics and arrangements of the microtubular cytoskeleton exhibit severe defects in cell functionality (Bao et al, 2001; Bichet et al, 2001; Burk et al, 2001; Ishida & Hashimoto, 2007; Ishida et al, 2007; Samakovli & Komis, 2017; Panteris et al, 2018). Plant hormones and their complex regulatory networks steer all aspects of plant growth and development (Petricka et al, 2012), among which auxin and cytokinin are key hormonal regulators of cell division and differentiation. Both hormones are required to maintain the proliferative activity of cells in suspension cultures (Skoog & Miller, 1957). In planta, the auxin–cytokinin crosstalk has a crucial morphogenetic function in the post-embryonic initiation and formation of new organs, such as lateral roots, shoots, leaves, or flowers, as well as in the control of the organization and activity of shoot and root apical meristems (Dello Ioio et al, 2008; Ruzicka et al, 2009; Marhavý et al, 2011; Chandler & Werr, 2015; Schaller et al, 2015). The interplay of auxin and cytokinin in the regulation of the root patterning is well described. The ratio of the auxin-to-cytokinin activities along the longitudinal root growth axis determines the cellular progression through distinct phases of the cytogenesis, thereby defining the size of the meristematic zone, the timing and dynamics of cell transition to elongation and the differentiation phase (Billou et al, 2005; Dello Ioio et al, 2008; Ruzicka et al, 2009; Takatsuka & Umeda, 2014; Di Mambro et al, 2017). However, the contribution of auxin and cytokinin in the regulation of the microtubular cytoskeleton activity associated with the cell cytogenesis is scarcely understood. Recently, rapid auxin-triggered rearrangements of the MT network in the root epidermal and hypocotyl cells have been reported (Nick et al, 1992; Takesue & Shibaoka, 1998; Takahashi et al, 2003; Le et al, 2005; Vineyard et al, 2013; Chen et al, 2014; True & Shaw, 2020). In addition, auxin has been proposed to coordinate reorganization of the cytoskeleton in the pericycle and endodermis during early phases of the lateral root organogenesis (Vilches Barro et al, 2019). Whereas the auxin-mediated reconfiguration of CMTs in hypocotyls seems to be an indirect consequence of the enhanced cell expansion, the direct auxin effect on the CMT cytoskeleton has not been excluded in roots (Adamowski et al, 2019). Unlike the auxin interaction, that of cytokinin with CMT cytoskeleton has not been assessed thus far. Here, we demonstrate that the cytokinin pathway plays a role in the fine-tuning of the CMT arrangements and dynamics during cytogenesis. In Arabidopsis thaliana roots, a gradual increase of the cytokinin activity along the root longitudinal axis correlates with altered dynamics of the CMT cytoskeleton in root epidermal cells. Modulation of the cytokinin activity gradient by either cytokinin supply or modulation of cytokinin perception and signaling dramatically affects the dynamics of the MT cytoskeleton and interferes with the auxin-driven rearrangements of CMTs. However, compared to the rapid auxin effects, the cytokinin-mediated reconfiguration of CMTs is slower, suggesting that auxin and cytokinin might target different pathways that regulate the microtubular cytoskeleton activity. This is further supported by the observation that cytokinin affects MTs in animal cells as well, whereas the auxin effect is restricted to the regulation of plant MTs. However, whether cytokinin interferes with MTs through a regulatory pathway that might be partially evolutionarily conserved between animal and plant kingdoms remains to be resolved. Results Orientation and dynamics of CMTs change along the longitudinal root growth axis Root growth results from the steady production of new cells at the root apical meristem and from their gradual expansion. As cells exit the meristematic zone, they proceed through the transition zone, where they lose their proliferation capacity before undergoing a rapid expansion and differentiation (Baluška et al, 2010; Schaller et al, 2015) (Fig 1A). Throughout all the growth phases, CMTs have an essential function as a framework for the coordinated deposition of new cell wall material and cell shape maintenance (Elliott & Shaw, 2018). Although the core functions of the microtubular cytoskeleton across various cell types and phases of the cytogenesis are largely conserved, CMTs are highly dynamic and their activity is fine-tuned to accommodate plasticity of cell growth. CMTs in cells of different growth zones have distinct patterns, indicating that during the transition from the proliferative to the differentiation phase CMTs might undergo robust rearrangements (Sugimoto et al, 2000; Le et al, 2004; Oda, 2015). To capture the dynamics of the CMT network in the course of cell growth, we used a live track imaging of root epidermal cells using the vertical-stage confocal microscopy (Movie EV1), thus avoiding perturbations caused by gravity-induced changes in roots. Figure 1. Monitoring of cortical microtubules (CMTs) in root epidermal cells Root tip of Arabidopsis with distinct growth zones marked: meristematic (MZ, green), transition (TZ, blue), elongation (EZ, pink), and differentiation (DZ, yellow). Double arrow indicates the first expanded cortex cell considered as a start point of the TZ, which encompasses epidermal cells smaller than 45 μm. Epidermal cells reaching a length of more than 45 μm prior termination of the elongation are in the EZ. Scale bar 25 μm. CMTs visualized by the MAP4-GFP reporter (left) and scheme of the CMT orientations (right) in epidermal cells of distinct growth zones (TZ, EZ, and DZ). Individual cells monitored at two time points (0 and 60 min). Dark blue arrows mark the cell expansion direction. Scale bar 10 μm. Histograms of the CMT orientation distributions. Orientation was measured as an angle between CMTs and the longitudinal root growth axis, with 0°, 45°, and 90° corresponding to longitudinal, oblique, and transversal orientations, respectively. The proportion of CMTs in a certain orientation is calculated per cell (n = 15–20 cells per root growth zone with six–nine roots in four independent replicates). Analysis of the CMT plus-end growth with the EB1b-GFP reporter. CMT plus-end growth rates (μm/min) measured by tracking the EB1b-GFP marker for 20–30 min in epidermal root cells of three growth zones. In the boxplots, the center lines show medians; box limits indicate the 25th and 75th percentiles as determined by the GraphPad software; whiskers span minimum to maximum values; and individual data points are represented by dots. **P < 0.01, ****P < 0.0001 by Student's t-test. The number of CMT plus-end events (EB1b-GFP) tracked per cell was 20–100/min, of which the growth rate average is represented as a single dot; three–five cells per root growth zone, three–five roots analyzed per biological replicate in three independent experiments. On the right, single trajectories of EB1b-GFP signal tracked over 60 s in root epidermal cells at different growth zones. Scale bar 0.5 μm. Z-stack maximum image projection of the EB1b-GFP plus-end reporter tracked over 30 s, in epidermal cells at different growth zones. Scale bars 10 μm. CMTs visualized by MAP4-GFP in root epidermal cells at the TZ, the EZ, and the DZ prior (0) and 60 min after treatment with 1 μm oryzalin. Scale bar 10 μm. Quantification of the MAP4-GFP CMT reporter signal in wild-type root epidermal cells at the TZ, the EZ, and the DZ treated with mock (DMSO, white box), oryzalin (1 μM, light gray box), and cytokinin (CK; 10 μM 6-benzylaminopurine, BAP) and oryzalin (1 μM) (dark gray box). For double treatments, roots pretreated for 60 min with CK prior to transfer to medium supplemented with both compounds. Boxplots represent ratio between mean fluorescence intensity (arbitrary units) measured in epidermal cells at 60 and 0 min. The center lines show the medians, and the box limits indicate the 25th and 75th percentiles; whiskers span the minimal to maximal values, and individual data points are represented by dots. Ratio close to 1 (segmented line) corresponds to the unchanged MAP-GFP signal for 60 min (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Student's t-test, n = 3–10 cells per root growth zone with five–eight roots per condition in four independent replicates). Download figure Download PowerPoint To correlate the CMT activity with distinct phases of cell growth, we defined root zones based on criteria described in previous reports (Verbelen et al, 2006; Ivanov & Dubrovsky, 2013; Slovak et al, 2016; Pavelescu et al, 2018). In the transition zone (TZ), cells start to elongate until they reach a length of ~ 45 μm; the elongation zone (EZ) includes cells longer than 45 μm until they enter the differentiation zone (DZ) characterized by initiation of root hairs (Fig 1A). To minimize variability, all analyses of CMTs were done in the atrichoblast cell file, i.e., epidermal cells that do not form root hairs, unless mentioned differently (Fig EV1A). To visualize CMTs, we employed the microtubule reporters MAP4-GFP (Marc et al, 1998); mCherry-TUA5 (Gutierrez et al, 2009); and immunocytochemistry with α-tubulin-specific antibodies. The Fiji software (https://fiji.sc/) was applied to quantify orientation by scoring the proportions of CMTs in certain orientations. In our assays, transversal and longitudinal orientations corresponded to an angle of 90° and 0° between CMTs and the longitudinal root growth axis, respectively (Fig 1C). Click here to expand this figure. Figure EV1. Monitoring of cortical microtubules (CMTs) in root epidermal cells A. Top surface view of an Arabidopsis thaliana root. Cell walls were stained with propidium iodide. Trichoblast root epidermal cells are marked in purple. Scale bar 25 μm. B. Immunostaining of α-tubulin in epidermal cells of the transition zone (TZ) and the elongation zone (EZ) of wild-type roots after 60 min of treatment with mock (DMSO), cytokinin (CK, 10 μM BAP), auxin (0.1 μM NAA), or CK and auxin. For the double CK and auxin treatment, roots were pretreated for 60 min with cytokinin and then transferred to medium supplemented with both compounds. Histograms present the CMT orientation distributions (%) in epidermal cells of the TZ and the EZ treated as indicated. n = 10–24 cells per growth zone in five–eight roots per condition were analyzed. Scale bar 10 μm. C–G. EB1b-GFP plus-end trajectories tinted with a color code according to the speed of their growth rates. The EB1b-GFP plus-end trajectories were monitored for 5 min in epidermal root cells and were analyzed by TrackMate plug-in (Fiji) at the elongation zone (EZ) (C, E–G) and at the differentiation zone (DZ) (D) after 60 min of treatment in mock (DMSO) (C, D), CK (10 μM BAP) (E), auxin (0.1 μM NAA) (F), or CK and auxin (10 μM BAP and 0.1 μM NAA) (G). For the double CK and auxin treatment, roots were pretreated for 60 min with CK prior to transfer to medium supplemented with both compounds. Scale bar 10 μm. H. Monitoring of CMTs in epidermal cells of the EZ using 35S::mCherry-TUA5 marker. Roots were incubated for 1 h in mock (DMSO) or CK (BAP 10 μM) supplemented medium. Five minutes of time-lapse videos and 20 μm segment of the cell (yellow dashed lines) were used to perform Kymograph analysis (upper panels), which were quantified by KymoButler software (lower panels with single trajectories included in the quantification are colored and numbered). Average track velocities (μm/min) are represented by boxplots. The center lines show medians; box limits indicate the 25th and 75th percentiles as determined by the GraphPad software; whiskers span minimum to maximum values; and individual data points are represented by dots. ****P < 0.0001 by Student's t-test. n = 3 biological replicates per condition, total number of trajectories analyzed n = 127 and n = 118 for mock- and CK-treated roots, respectively. I, J. Analysis of CMTs (I) and root growth (J) recovery after CK treatment. Roots of 5-day-old seedlings were incubated in mock (DMSO; upper panel) or CK (BAP 10 μM; lower panel) containing medium for 1 h and transferred to mock medium (I, J). Time-lapse images capture CMTs visualized by MAP4-GFP in epidermal root cells at the EZ at 0, 1, 2, 2.5, 3, and 5 h after transfer. Schemes of CMT distribution and cell length at every time next to the images. Scale bar 10 μm (I). Relative root growth (mm) measured during 555 min. Seedlings incubated in mock and transfer to mock medium (black line), pretreated with CK and transferred to mock medium (gray line), pretreated with CK and transferred to CK containing medium (blue line). Mean ± s.d.; n = 10–18 roots per condition. Download figure Download PowerPoint In epidermal cells of the TZ and the EZ, CMTs were arranged transversally (84.42 ± 1.1° and 84.45 ± 0.9°, respectively) and their orientation remained unchanged for 60 min. In the DZ, the cellular elongation growth ceased and the CMTs changed from a transversal to an oblique orientation, reaching an angle ~ 51.55 ± 1.5° (Fig 1B and C; Movie EV1; Table EV1). Although the MAP4-GFP marker is an excellent tool to visualize CMT cytoskeleton, it has limitations due to the chimeric origin of the microtubular binding part of the protein (Marc et al, 1998). Importantly, visualization of CMTs with α-tubulin-specific antibodies corroborated patterns of CMTs in distinct root growth zones as detected with the MAP4-GFP reporter (Fig EV1B). The CMT network is highly dynamic and individual CMTs alternate between growing and shortening phases, enabling their quick assembly and disassembly (Horio & Murata, 2014). To gain insights into dynamics of CMTs in distinct root growth zones, we used the EB1b-GFP marker to monitor growth rate at plus-end of CMTs (Buschmann et al, 2010; Wong & Hashimoto, 2017). By means of spinning disk microscopy, the EB1b-GFP signal was followed for 20–30 min and the CMT growth rates were extracted from time-lapse image sequences. In the epidermal cells at the TZ and the EZ, CMTs plus-end growth rates reached average values of 2.6 ± 0.07 and 2.4 ± 0.07 μm/min, respectively (Fig 1D, Movie EV2). In cells of the DZ, the CMT plus-end growth rate was significantly lower (1.8 ± 0.08 μm/min) than that of less differentiated cells at the TZ and the EZ (Fig 1D, Movie EV3), suggesting that the growth of CMTs at plus-end might cease in cells undergoing differentiation. Visualization of the growth trajectories of CMTs at plus-end by the maximum projection of EB1b-GFP monitored for 30 s supports the transversal orientation of CMTs in cells at the TZ and the EZ, but slanting in the DZ, in line with the MAP4-GFP pattern (Figs 1E and EV1C and D; Movies EV2 and EV3). To explore whether the dynamics of CMTs adapt to the cellular differentiation status, we tested the CMT sensitivity to oryzalin. This drug sequesters free dimers of tubulin and prevents their addition to the CMT plus-ends, thereby triggering the rapid depolymerization of CMTs (Morejohn & Fosket, 1991; Hugdahl & Morejohn, 1993). It has been shown that CMTs stabilized by taxol are only partially sensitive to oryzalin (Morejohn et al, 1987; Hugdahl & Morejohn, 1993). Thus, we hypothesized that differences in the CMT dynamics, as suggested by analyses of microtubule growth rate at plus-ends in cells of distinct root zones, might be manifested by changed sensitivity of CMTs to oryzalin. Oryzalin applied for 60 min led to a quick depletion of CMTs in epidermal cells at the TZ and the EZ, but in cells of the DZ, CMTs were largely insensitive to oryzalin (Fig 1F and G), hinting at changed dynamics of CMTs in cells of the DZ when compared to the TZ and the EZ. Hence, as cells progress through distinct root growth zones, the CMT network undergoes a pronounced reconfiguration. Transversal positioning and enhanced growth rate at plus-end of CMTs might provide optimal arrangements for the effective deposition of cell wall components in growing cells at the TZ and the EZ when compared to CMTs in differentiated cells, in which the reduced plus-end growth rate and reorientation of CMTs to an oblique orientation correlate with termination of cell expansion. Cytokinin and auxin form distinct response gradients along the longitudinal axis of the Arabidopsis root Coordinated, spatio-temporally controlled transition of root cells through the proliferation and expansion phases until the fully differentiated stage is acquired defines the overall kinetics of the primary root growth (Verbelen et al, 2006; Dello Ioio et al, 2008; Petricka et al, 2012). Auxin and cytokinin are among essential endogenous regulatory molecules, of which the mutually antagonistic activities at the distal root tip have been shown to control the balance between the cell proliferation rate and the transition to elongation and differentiation (Dello Ioio et al, 2008; Moubayidin et al, 2009; Petersson et al, 2009; Antoniadi & Pla, 2015; Di Mambro et al, 2017). The distinct patterns and dynamics of CMTs detected in cells of the different root growth zones prompted us to thoroughly monitor the activity of the auxin and cytokinin pathways along the longitudinal root growth axis. To closely examine the balance between the auxin–cytokinin responses in individual cells, we used the novel biosensor TCSn::ntdT:tNOS-DR5v2:3nGFP (Smet et al, 2019). In agreement with previous reports, we detected mutually complementary, partially overlapping expression patterns the sensitive reporters of auxin (DR5v2:3nGFP) and cytokinin (TCSn::ntdT:tNOS) in the provasculature, stem cell niche, columella, and lateral root cap (Bishopp et al, 2011; Bielach et al, 2012; Zürcher et al, 2013; Sozzani & Iyer-Pascuzzi, 2014) (Fig 2A). Expression analyses of DR5v2:3GFP and TCSn::ntdT:tNOS in epidermis along the root growth axis revealed distinct response patterns of these two hormonal pathways (Fig 2A and B). While increase of DR5v2:3nGFP along the root axis followed a relatively shallow gradient, the TCSn::ntdT:tNOS expression profile exhibited gradual increase across the TZ and the EZ toward the DZ (Fig 2A and B). Noteworthy, profiles of auxin and cytokinin responses in the epidermal cell files that give rise to root hairs (trichoblasts) differed from those observed in non-root hair cells (atrichoblasts) (Fig EV2A–C). Whereas in atrichoblasts the increase in cytokinin responses could be detected early after the transition into the differentiation phase, in neighboring trichoblasts the auxin responses prevailed and the cytokinin responses increased only after the root hairs were fully formed (Fig EV2A–D). Thus, analyses of the TCSn::ntdT:tNOS-DR5v2:3nGFP reporter confirmed the previously reported pattern of the auxin and cytokinin activities at the root apical meristem (Bielach et al, 2012) and revealed that the transition from proliferation to differentiation in epidermal cells is accompanied with gradual enhancement of cytokinin activity. Figure 2. Cytokinin modulates CMT patterns and dynamics A. Arabidopsis root expressing the dual reporter TCSn::ntdT:tNOS-DR5v2::3nGFP that is sensitive to cytokinin (CK) and auxin. CK (red), auxin (green), and overlay of both signals detected in nuclei of cells at the root tip (top, scale bar 50 μm). Green, pink, and blue arrowheads point at epidermal cells located in the meristematic zone (MZ), transition zone (TZ), and elongation zone (EZ), respectively. Magnification of the TZ shown (bottom, scale bar 10 μm). B. Relative fluorescence intensity of TCSn::ntdT:tNOS signal (red) and DR5v2:3nGFP signal (green) measured in epidermal cells along the longitudinal root growth axis. Cell number 1 corresponds to a meristematic cell placed at position −4 before the beginning of TZ (as marked by green arrow at 2A). Mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Student's t-test, n = 18 roots. C–F. CMTs visualized with MAP4-GFP in root epidermal cells of the TZ and the EZ at time points 0 and 60 min after CK (C), CK and oryzalin (D), auxin (E), and CK and auxin (F) treatments, and in DZ cells at time points 0 and 60 min after auxin treatment (E). As CK and auxin sources, 10 μM BAP and 0.1 μM NAA were used, respectively, and 1 μM oryzalin. For the double (CK/oryzalin and CK/auxin) treatments, roots were pretreated with CK for 60 min prior to transfer to medium supplemented with both compounds. Scale bar 10 μm. G. Histograms of CMT orientation distributions in epidermal cells of the TZ and the EZ at time points 0 and 60 min after hormonal treatments as described above (C, E and F). n = 15–20 cells per root growth zone with five–eight roots per condition in four independent replicates. H. Analysis of the CMT plus-end growth with the EB1b-GFP reporter. Z-stack maximum image projections of EB1b-GFP tracked over 30 s in epidermal cells without (mock, DMSO) and with CK, auxin, or CK and auxin treatment (60 min). Scale bars 10 μm. I. CMT plus-end growth rates (μm/min) measured by tracking of EB1b-GFP reporter over 20–30 min in epidermal cells of diffe
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