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First published online November 19, 2004; 10.1104/pp.103.031591 Plant Physiology 136:3905-3919 (2004) © 2004 American Society of Plant Biologists Reorganization and in Vivo Dynamics of Microtubules during Arabidopsis Root Hair Development[w]Laboratory for Biochemistry and Molecular Cytology, Ghent University, 9000 Gent, Belgium (N.V.B., P.V.O.); and Department of Biological Sciences, Macquarie University, Sydney, New South Wales 2109, Australia (G.J.)
Root hairs emerge from epidermal root cells (trichoblasts) and differentiate by highly localized tip growth. Microtubules (MTs) are essential for establishing and maintaining the growth polarity of root hairs. The current knowledge about the configuration of the MT cytoskeleton during root hair development is largely based on experiments on fixed material, and reorganization and in vivo dynamics of MTs during root hair development is at present unclear. This in vivo study provides new insights into the mechanisms of MT (re)organization during root hair development in Arabidopsis (Arabidopsis thaliana). Expression of a binding site of the MT-associated protein-4 tagged with green fluorescent protein enabled imaging of MT nucleation, growth, and shortening and revealed distinct MT configurations. Depending on the dynamics of the different MT populations during root hair development, either repeated two-dimensional (x, y, t) or repeated three-dimensional (x, y, z, t) scanning was performed. Furthermore, a new image evaluation tool was developed to reveal important data on MT instability. The data show how MTs reorient after apparent contact with other MTs and support a model for MT alignment based on repeated reorientation of dynamic MT growth.
A root hair is a long, lateral, tubular extension of an epidermal root cell formed by a process called tip growth. Diffuse longitudinal growth of the epidermal cell undergoes transition to highly localized and polarized growth at one specific site. After the formation of an initial bulge, the root hair grows by polarized exocytosis and deposition of cell wall material confined to the tip (Schnepf, 1986  ). In an early phase, 3-d-old Arabidopsis (Arabidopsis thaliana) root hairs grow out at a rate of 0.4 µm/min, followed by a late growth phase at 1 to 2.5 µm/min (Dolan et al., 1994). Tip growth is characterized by the presence of a vesicle-rich region, a reverse-fountain type of cytoplasmic streaming, and a gradient of cytoplasmic Ca2+ toward the hair tip (Pierson et al., 1996; Galway et al., 1997; Miller et al., 1997, 1999; Wymer et al., 1997; de Ruijter et al., 1998; Yang, 1998). During growth arrest cytoplasmic streaming switches from reverse-fountain streaming to circular streaming, and this reorganization is coupled to the disappearance of the vesicle-rich region (Miller et al., 1997).
It has been accepted that root hair outgrowth is associated with microtubule (MT) and actin filament reorientations (Emons and Derksen, 1986
Filamentous actin plays a fundamental role in the tip growth process (for review, see Geitmann and Emons, 2000
Recent in vivo studies have provided new insights into MT dynamics in plant cells. Sieberer et al. (2002)
In general, reorganization can occur by polymerization of new MTs, reorientation of intact MTs, or polymerization of existing MTs in a new direction (Kropf et al., 1998
Another feature of MT dynamics adds to the complexity of MT reorganization. MTs are capable of rapid rearrangement by subunit exchange, which allows them to grow and shorten. These mechanisms have been thoroughly investigated in animal cells (for review, see Desai and Mitchinson, 1997
Transgenic Arabidopsis plants expressing a GFP-MBD of MT-associated protein-4 (MAP4) fusion protein (Marc et al., 1998
To allow direct in vivo observations of MTs, transgenic plants stably expressing GFP-MBD of MAP4 were used (Marc et al., 1998 ). Arabidopsis plants carrying this construct were phenotypically normal and displayed normal root hair formation. As the root and the surrounding medium were transferred as a whole to the small-growth chamber suitable for microscopy, the root hairs continued to grow for several hours during imaging. The mean root hair growth rate during imaging was approximately 1 µm/min. This is in agreement with data from literature (Dolan et al., 1994) and suggests that root hair development was not affected by imaging.
The cortical arrays in elongating epidermal cells formed helical arrays in which the MTs were oriented transversely or at a slightly oblique angle (60–90° to the long axis of the cell). At the onset of root hair outgrowth, the MTs in the trichoblasts reorganized, whereas the MTs in atrichoblasts stayed in transverse arrays (Fig. 1, A and B). In the apical region of the trichoblast (near the bulge) the MTs disorganized (Fig. 1, A and B). This was also observed in lettuce by Takahashi et al. (2003a)
Distinct Configuration Patterns of CMTs Versus EMTs in Elongating Root Hairs
MT configuration was followed in 4-D (x, y, z, t) during root hair expansion over long time periods. The 3-D time series images (x, y, z, t) were reduced to movies by projecting the maximum fluorescence of the confocal-optical sections for each time step to a 2-D image. In the cortical region an array with a high density of CMTs was observed (Fig. 2A; Supplemental Movies 3 and 4) similar to previous immunofluorescence studies (Lloyd et al., 1987
During further expansion of the root hair, the nucleus migrated into the tube and positioned at a more-or-less constant distance from the tip (Ketelaar et al., 2002 ). Figure 3A shows the situation after the nucleus has migrated into the root hair. Thick bundles of EMTs extended between the tip and the nucleus and formed an array around the nucleus. The cross section through the nucleus in Figure 3B clearly shows that the fluorescence was present at the edge of the nucleus and was most intense at the ends toward the tip and the base of the root hair. The EMT bundles branched in the tip and seemed to extend toward the CMTs in the subapical region. In highly vacuolated regions of the root hairs, the EMTs were localized close to the cortex as the endoplasm was reduced to a thin layer between the vacuole and the plasma membrane. In this study, the EMT bundles at the subcortex were still clearly distinguishable from the CMT array but sometimes difficult to represent in a single projection. Supplemental Movie 5 shows the dynamics in the core-central sections and at a more upper level (including part of the CMTs). The EMTs are very dynamic and continuously change their orientation and configuration. Possibly these movements are related to the reorganizations of the cytoplasmic strands. Close to the bundles, individual fluorescent spots could be distinguished with short MTs radiating from these spots. The spots are clearly visible in the subcortex (lower part of Fig. 4A) and in the endoplasm (Fig. 4B; Supplemental Movie 6). MTs extending from the nucleus toward the cortex are indicated by arrows in Figure 4C. The elongation and reorientation of single MTs were imaged by repeated 2-D images (x, y, t) with increased focal depth taken every second (Supplemental Movie 6).
The Organization Pattern of MTs in Elongating Root Hairs Is Different from the Pattern in Full-Grown Root Hairs
Since cytological structures rearrange during growth arrest, it was interesting to investigate whether these reorganizations coincide with MT rearrangements. One of the features visible during growth arrest was that the vesicle-rich region narrowed and finally completely disappeared in full-grown root hairs (compare Fig. 5, A, E, and I). The cytoplasmic streaming changed from reverse-fountain streaming to circular streaming, as described earlier by Miller et al. (1997)
There are three major configuration differences between growing and full-grown root hairs. First, and most obviously, there are fewer CMTs in full-grown root hairs compared to elongating root hairs (Fig. 5, B versus J). The growing root hairs had a high-density array of CMTs. In full-grown root hairs, CMTs had a longitudinal or helical organization (for review, see Geitmann and Emons, 2000 ); only very few MTs were transversely oriented. Second, the MTs in elongating root hairs didn't reach the very tip, whereas some MTs reached the very tip in growth-arresting root hairs and could even curve in the tip of mature root hairs (Fig. 5, B, F, and J, respectively). Third, the EMTs disappeared during growth arrest and there were no MTs extending between the nucleus and the tip in full-grown root hairs (compare Fig. 5, C, G, and K). Occasionally, short MTs were observed close to the nucleus in full-grown root hairs.
To determine whether MTs can reassemble independently from the interior organizing sites such as the nuclear surface, root hairs were depleted of MTs by the depolymerizing drug oryzalin. The MTs in the root hair and its cell body were completely depolymerized after a 10-min treatment with 1 µM oryzalin but not in the neighboring epidermal cells (Fig. 6A). It is nearly impossible to visualize the MT configurations within a single root hair before and after drug application because manipulation alters positioning of the root hair and observation has to start directly after treatment. However, all root hairs had normal MT configurations before treatment. Only 10 min after washing out the oryzalin drug, the first signs of recovery were observed. Single dense spots appeared at the cortex in the epidermal cell and in the root hair tube (Fig. 6A; Supplemental Movie 7). In the following minutes MT clusters were formed in the (sub)-cortex (Fig. 6A) and in the endoplasm (Fig. 6B) at the places where the dense spots first appeared. Some clusters were very similar to the branching clusters observed by Wasteneys and Williamson (1989a)
Dynamic Instability of MTs Revealed with Difference Imaging In the nondrug-treated cells a method was developed to better visualize the CMT dynamics. Each frame of the movie is compared with the following frame by combining the pair into a single color image. Each frame forms the red channel when it is the first of a pair (Fig. 7A) and the green channel when it is the second of a pair (Fig. 7B). The resulting color movie aids visualization of changes in the MT cytoskeleton over time (Fig. 7C). Fluorescence present only in the first image of each pair appears red; fluorescence present only in the second image of each pair appears green; fluorescence levels that remain constant from frame to frame will combine the red and green channels to appear yellow in the difference image (Fig. 7, C and D). The MTs of fixed cells lacking dynamics will appear completely yellow. Transmission images taken together with the fluorescence images served as control for possible specimen drift.
In elongating root hairs few MTs remained in the same position between two sequential time points at 1-min intervals. This reorientation may be caused by MT translocation or turnover. The fast reorientation together with the high-MT density makes it difficult to distinguish between these two possibilities. The intensity profiles along the root hair of those pixels in Figure 7C that are exclusively green or exclusively red are plotted in Figure 7E and give information about the net growth and shortening. The green peak close to the vesicle-rich region indicates formation of new MTs during root hair growth. This increase in fluorescence indicates either de novo MT formation in the new part of the root hair or extension of MTs from the old region into the new. On the other hand the red peak, representing MTs in the first frame that are absent in the second, indicates that MT density is reduced behind the region of newly appearing MTs, represented by the green peak. This suggests that most MTs that were formed close to the vesicle-rich region translocated toward the tip or depolymerized during the next minute. It was not possible to directly make the distinction between putative-bundled CMTs and individual CMTs due to the limited resolving power of the microscope. However, in full-grown root hairs several observations support the idea that dynamics of single CMTs were observed. First, CMTs were never observed to move laterally or merge to another CMT so that they could no longer be individually resolved; second, newly formed CMTs and existing MTs showed equal intensity; and finally the dynamic-instability data (see below) indicated that the CMTs were one continuous unit as complete shortening was observed. Therefore, as there was no indication that bundled CMTs occurred in full-grown root hairs, in the subsequent results the observed CMTs will be regarded as single CMTs.
The visualization method also proved very useful to analyze changes of the CMTs in full-grown root hairs. Different characteristics of the individual CMTs were revealed. In contrast to the CMTs in growing root hairs, most of the CMTs in full-grown root hairs are colored in yellow (Fig. 7H). There are no green or red changes along the yellow MTs, supporting the idea that the observed MTs in full-grown root hairs are single MTs (Supplemental Movie 9). This is in contrast with the green and red portions appearing along the yellow MTs in the transverse arrays of nonroot hair epidermal cells (data not shown), which suggests dynamics of multiple MTs lying along each other. In full-grown root hairs no MT displacement was observed in the z-direction (Fig. 8A; MT remains yellow); green and red portions were observed only at the MT ends. In addition, the majority of CMTs showed no lateral movements over time (Fig. 8, B and C). These two observations suggest that the CMTs were fixed to the cortex, corresponding to the observations of Shaw et al. (2003)
Another important feature that could be directly determined from the difference images is the amount of growth (green) and shortening (red) at the MT ends (Fig. 7, F–I). The dynamics of both MT ends of five CMTs are depicted as MT life history plots in Figure 7J. The plots demonstrate that the growth rate is lower than the shortening rate, corresponding with the data of Dhonukshe and Gadella (2003) and Vos et al. (2004). It should be noted that the plots may represent an underestimation of the real situation, especially at the transition points where growth switches to shortening and vice versa. In contrast to these dynamic (plus) ends, the opposite minus ends were characterized with slow (72%) or no (28%; total n = 146) shortening (Fig. 7J). Only limited examples of breakage in CMTs were recorded (less than 2%). Detailed analysis of the behavior of 174 CMTs in 10 different full-grown root hairs revealed that the majority of CMT plus ends were oriented toward the root hair tip (82% ± 9%). Only 11% ± 6% of the plus ends were oriented toward the base and 7% ± 5% were transversely oriented. The difference image Figure 7H showing shortening in red and growth in green revealed that fast shortening of MTs occurred over the whole length of the full-grown root hair. Shortening events at MT plus ends could result in complete depolymerization of CMTs (MT4 in Fig. 7J). MTs that did not show depolymerization at the minus end before complete depolymerization of the plus end recovered in the next minute from the original initiation site. On the other hand, MTs that showed slow depolymerization at the minus end did not recover after complete depolymerization (MT4 in Fig. 7J). This suggests that the MT initiation site is inactivated after minus-end depolymerization.
Inspection of the movies (e.g. Fig. 8, F and G; Supplemental Movie 9) revealed another striking feature. When a CMT plus end apparently contacted another CMT, the CMT partly or completely depolymerized. Repeated 2-D (x, y, t) imaging of restricted well-focused regions showed that shortening after apparent contact occurs after a pause of 30 to 60 s with no length change. To better quantify the significance of this observation the number of CMT shortening events and apparent contacts of a CMT plus end with another CMT were counted in seven cells (Table I). The data clearly show that most shortening events occurred after the leading plus end encountered another CMT (80% ± 9%). Only 20% ± 10% of the shortenings occurred without apparent contact. Apparent contacts of plus ends with other CMTs could also result in crossings without shortening, but this occurred at a much lower frequency (27.5% ± 5%). In addition approximately 50% of this type of apparent contact occurred with transversely oriented CMTs (Table I), which were markedly shorter in length than the other more longitudinally oriented MTs. The data in Table I represent only the dynamic events during imaging. Only those crossing events that occurred during imaging were counted, as also the previous apparent contacts could not be taken into account. As the time gap between the images is 1 min, the growth or shortening within this period cannot be visualized. Due to this limited-time resolution, the dynamics of the MTs may be underestimated; e.g. it is possible that the growth and subsequent rapid shortening taking place between two time points is seen as a net shortening. In this respect, the number of events within the class of shortenings without apparent contact might be overestimated. However, as there is a pause of 30 to 60 s with no change in length after encountering another MT, the chance to observe an MT in an apparent contact state is high, and the chance that growth, apparent contact with pause, and subsequent rapid shortening occurs completely within the timeframe of 1 min (observed as a shortening without an apparent contact) can be expected to be low.
Interestingly, those CMTs that recovered after shortening (n = 64) all grew back in a slightly different direction. In the extra sections of Figure 8, F and G two examples are depicted where an MT in this way showed three subsequent different orientations during one movie of 30 min. The apparent contacts followed by shortening are indicated by yellow triangles in Figure 7J, and the deviation in growth direction ranged from 2° to 11° (see figure legend Fig. 7J). These observations suggest that the reorientation of a CMT after apparent contact is part of a mechanism for MT alignment. To test this hypothesis, the frequencies of shortening events were observed in root hairs with different densities of CMTs. In a root hair with fewer CMTs (Table II, root hairs 1 and 2), significantly fewer shortening events occurred over time than in root hairs with more CMTs (root hair 3). One would expect a shorter CMT length if more shortening events occur. Indeed, the CMT lengths were significantly shorter in root hair 3 compared to root hairs 1 and 2 (Table II). Finally, the CMTs in root hairs 1 and 2 are more uniformly aligned than in root hair 3 (Table II). Presumably, root hair 3 was more recently arrested in growth, showing more CMTs but less well aligned CMTs. Together with the fact that CMTs grew in a slightly different direction after shortening (Fig. 8, F and G) and the fact that some CMTs completely depolymerized after apparent contact, this process may finally result in fewer, longer, and better aligned CMTs (root hairs 1 and 2). Data modeling for the different parameters will be performed in the future to investigate if this correlation is real.
MT Reorganization Precedes Change of Growth Direction
At the onset of root hair outgrowth MTs disorganize in Arabidopsis in trichoblasts, in contrast to the transverse arrays in the atrichoblasts (Fig. 1A). Similar results have been reported for maize and lettuce (Balu
Reorganization of MTs during root hair development is in part the result of polymerization of new MTs. MT initiation sites were observed as discrete spots with radiating MTs at the onset of root hair outgrowth and near the EMTs during root hair growth. MTs also initiate at the (sub)-cortex in full-grown root hairs, but the initiation sites are not distinct from the MTs. Due to the dense CMT array in growing root hairs it is impossible to see if there are discrete spots. MTs in the newly formed tip are continuously added during growth. If fluorescent spots indicate new initiation sites of MT formation, there is no evidence that new MTs are formed near the vesicle-rich region. Therefore, MTs in the region near the vesicle-rich region have to be extensions of MTs from the old region. At present there are different views about the origin of the CMTs. One model suggests that all MTs are nucleated at the nuclear membrane and then transported (overview in Lambert and Lloyd, 1994
This in vivo study demonstrated EMTs for the first time, to our knowledge, in Arabidopsis root hairs. EMTs could not be demonstrated by immunofluorescence previously (Ketelaar et al., 2002
Before nuclear migration perinuclear EMTs in Arabidopsis growing root hairs extend from the nucleus toward the tip. During nuclear migration the perinuclear EMTs form a very tight array surrounding the nucleus and extend as bundles in the subapical region toward the tip. In M. truncatula root hairs EMTs are thought to configure the subapical region and to be necessary to keep the nucleus close to the root hair tip (Sieberer et al., 2002
CMT dynamics in full-grown root hairs was analyzed in detail using difference images. CMTs showed no lateral or z-direction movement, except if newly formed. Possibly, CMTs became anchored to the cortex by specific MAPs during maturation (for review, see Lloyd and Hussey, 2001 Quantitative results revealed that the plus ends of CMTs seemed to shorten due to other CMTs in their path (Table I). Shortening events mainly occurred (80%) when the plus end of one CMT encountered another CMT (Table I). Since two populations of CMTs were observed, those that appear to be anchored to the plasma membrane and those located at a slightly different level in the subcortex, this may explain the apparently different behavior following an apparent contact. If the CMTs are both anchored to the plasma membrane, then a contact is likely to be real. Otherwise the two CMTs are likely to just cross the line of sight at different levels. Transverse CMTs are likely to be positioned at a slightly different level in the cortex and cross with other CMTs rather than collide. Transverse MTs are shorter in length and might not yet be anchored to the cortex, which can explain their different positioning. The feature of shortening after contact is probably more obvious in plant cells than in animal cells, as dense arrays of CMTs are confined to a thin cortical layer in plant cells.
One possible explanation for shortening after apparent contact is based on force in encountering an obstacle. There is evidence for this in vitro (Janson et al., 2003
It is clear that reorganization is a distinct process from initiation of the MTs, as MTs first appear as disorganized structures and order later (this study, Fig. 6; Wasteneys and Williamson, 1989b
From these results a touch-and-reorientation model is suggested in which CMTs first grow in arbitrary directions and reorient after contact with other MTs, finally resulting in the alignment of CMTs. When the MTs are less dense, the CMTs are less likely to shorten and more likely to grow undisturbed (Table II). In full-grown root hairs, this mechanism is observable as CMT density is low and dynamics relatively slow. It is possible that this type of reorientation also occurs in growing root hairs, but may be difficult to observe as the distance between the CMTs is small, which can result in reorientation at a much faster rate. During root hair growth CMTs have to follow the tip and are presumably mainly ordered in a longitudinal direction toward the root hair tip. Possibly there is a link between ion gradients at the apical root hair tip and the MTs. Bibikova et al. (1999)
Plant Material and Growth Conditions
For labeling of the MTs a construct of the GFP-MBD-chimeric gene, kindly supplied by Richard Cyr (Biology Department, Pennsylvania State University) described by Marc et al. (1998)
A series of 3-D images (x, y, z) were recorded with a confocal laser-scanning microscope (Radiance 2000, Bio-Rad, Hertfordshire, UK), mounted on an Eclipse 300 Nikon microscope (Tokyo). A 40x S Fluor oil (NA 1.30) or 60x Plan Apo (NA 1.2) water immersion lens was used. Enhanced GFP was excited with a 488-nm line of an Argon Ion laser and detected with an HQ 528/50 nm emission filter. Simultaneous transmission images were taken to visualize the cytoplasmic architecture. A typical 3-D stack (x, y, z) consisted of 25 optical sections of whole root hairs or parts of root hairs with a separating distance of 1 µm between the successive sections. Time lapse images were recorded automatically. Either 3-D (x, y, z) optical sections were imaged every minute resulting in 4-D datasets (x, y, z, t) or 2-D single optical sections (x, y) were imaged every second (x, y, t). The 3-D (x, y, z) optical sections were imaged with a pinhole of 1.9 airy disc units, whereas the single optical sections (x, y) were imaged with a larger pinhole of 3 airy disc units (except Supplemental Movie 2).
The MT-depolymerizing drug oryzalin (Sigma-Aldrich) was dissolved in dimethyl sulfoxide (Merck, Darmstadt, Germany) as a 10-mM stock solution and diluted in a phosphate buffered solution to a final concentration of 1 µM. The final dimethyl sulfoxide concentration did not exceed 0.01%, and this concentration had no effect on the MTs. The roots were treated by supplementing the drug solution to the Phytagel for 10 min. The drug was washed away by applying and removing buffered saline solution to the phytagel three times.
All image analysis was performed with the public domain NIH ImageJ program (developed at the United States National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/ij) together with a suite of purpose-written plugin programs and macros. Plugins were written for the reduction of the 3-D time lapse datasets to movies and for tracking of changes between images. They are available on request from the authors. The growth and shortening patterns of CMTs could easily be measured on the difference images (see above "Results" section; shortening = red, growth = green) in combination with the reslice tool on the movie stack. By drawing a line along the maximal length of the MT, the reslice tool gave an output of the lengths of the MTs, with the shortening and growth indicated in red and green, respectively. The density of CMTs was measured by drawing lines at well-defined distances perpendicular to the root hair axis. The plot profile of the intensities together with visual inspection made it possible to count the number of CMTs. The number of shortening events was measured by counting the significant red lines in the difference images. Root hair lengths were measured on transmission images. All movies were converted to the avi format using ImageJ. The t test (0.05) was used to measure significant differences in frequency of shortening for the different root hairs. Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third-party owners of all or parts of the material. Obtaining any permission will be the responsibility of the requestor.
We thank Richard Cyr (Biology Department, Pennsylvania State University) for the MAP4 construct and Karimi Mansour (Flemish Interuniversity Institute for Biotechnology, Ghent University) for establishing the stable transformed Arabidopsis lines. We are grateful to Danny Geelen (VIB, Ghent University) for helpful comments on the manuscript; Björn Sieberer for discussions; and Alejandro Calderon-Urrea (California State University, Fresno), Nico Thooft, and Els Van Damme (Department of Molecular Biotechnology, Ghent University) for critical reading of the text. Received August 12, 2003; returned for revision June 30, 2004; accepted July 21, 2004.
1 Present address: In Vitro Fertilization Center, Center for Radio-Immunology, Industriepark 3b, 9052 Zwijnaarde, Belgium. 
[w] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.031591. * Corresponding author; e-mail nvanbruaen{at}cri.be; fax 32–9–264–62–19.
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ABSTRACT
RESULTS
-tubulin gene expression causes formation of multiple root hairs by single epidermal cells and ectopic root hair formation in tissues that are normally root hair free. This was the first evidence, to our knowledge, for a function of the MT cytoskeleton during root hair initiation, in particular suggesting a role for MT disassembly (caused by low 
-tubulin complexes located at the spindle pole body in yeast or at the centrosome in animal cells (for review, see Schiebel, 2000
alaj J, Mathur J, Braun M, Jasper F, 
