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First published online February 11, 2005; 10.1104/pp.104.052613 Plant Physiology 137:1027-1036 (2005) © 2005 American Society of Plant Biologists Regulation of Secondary Cell Wall Development by Cortical Microtubules during Tracheary Element Differentiation in Arabidopsis Cell Suspensions1,[w]Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba 277–8562, Japan (Y.O., S.H.); and Department of Biology, Faculty of Sciences, Kobe University, Nada-ku, Kobe 657–8501, Japan (T.M.)
Cortical microtubules participate in the deposition of patterned secondary walls in tracheary element differentiation. In this study, we established a system to induce the differentiation of tracheary elements using a transgenic Arabidopsis (Arabidopsis thaliana) cell suspension stably expressing a green fluorescent protein-tubulin fusion protein. Approximately 30% of the cells differentiated into tracheary elements 96 h after culture in auxin-free media containing 1 µM brassinolide. With this differentiation system, we have been able to time-sequentially elucidate microtubule arrangement during secondary wall thickening. The development of secondary walls could be followed in living cells by staining with fluorescein-conjugated wheat germ agglutinin, and the three-dimensional structures of the secondary walls could be simultaneously analyzed. A single microtubule bundle first appeared beneath the narrow secondary wall and then developed into two separate bundles locating along both sides of the developing secondary wall. Microtubule inhibitors affected secondary wall thickening, suggesting that the pair of microtubule bundles adjacent to the secondary wall played a crucial role in the regulation of secondary wall development.
Tracheary elements, known as components of xylem vessels, are equipped with elaborately patterned secondary cell walls under the primary cell walls, which give rise to their characteristic appearances. In contrast with primary cell walls, secondary walls are comparatively thick and stout due to their high cellulose content and their lignin and hemicellulose deposits, and provide the tracheary elements with enough strength to withstand high negative pressure within the vessels (Ye, 2002  ).
Cortical microtubules are known to participate in the regulation of secondary wall deposition. The microtubules lying directly beneath the secondary walls have been observed for over four decades by transmission electron microscopy in several plant species (Hepler and Newcomb, 1964
The differentiation of tracheary elements can be induced semisynchronously in zinnia (Zinnia elegans) mesophyll cells (Fukuda and Komamine, 1980a
Previous studies of microtubules and secondary walls have been restricted to observations in chemically fixed cells. Therefore, the sequence of microtubule structures during secondary wall thickening could not be investigated in detail. Recently, observation of microtubules by the expression of green fluorescent protein (GFP)-tubulin fusion proteins, especially in stable transformants, has enabled time-lapse observations of microtubules in living cells and revealed several novel structures, dynamics, and functions of microtubules (Yoneda et al., 2003
The establishment of stable transformants of zinnia has not been reported, and there seem to be inherent difficulties in obtaining stable zinnia transformants. On the other hand, stable Arabidopsis transformants are easily obtainable. The use of Arabidopsis suspension cells will allow precise observations of microtubules in living cells. Furthermore, several genes involved in xylem development have already been identified in Arabidopsis mutants (Ye et al., 2002 For the reasons described above, we attempted to develop a tracheary element differentiation system using Arabidopsis cell suspensions. We first established a transgenic Arabidopsis cell suspension stably expressing a GFP-tubulin fusion protein and succeeded in differentiating them into tracheary elements. Based on the results, the roles of microtubules in secondary wall thickening are discussed.
Transformation of Arabidopsis Col-0 Cell Suspensions
To observe microtubules in living cells, we transformed Arabidopsis Col-0 cell suspensions with a vector containing a GFP(S65T)-tubulin fusion gene (Kumagai et al., 2001
Establishment of the Differentiation System of Tracheary Elements from AC-GT13 Cells
To establish an efficient system of tracheary element differentiation from AC-GT13 cells, the cells were transferred into various media containing different concentrations of phytohormones, such as auxin, cytokinin, and brassinosteroid, which were thought to be related to tracheary element differentiation (Fukuda, 1997
The effects of 2,4-D and BL were investigated further. The tracheary element differentiation rates increased with increasing concentrations of BL up to 1 µM (Fig. 2D), while an opposite tendency was observed for 2,4-D (Fig. 2E). This clearly demonstrated an inhibitory effect of 2,4-D on tracheary element differentiation, and therefore the 2,4-D was removed by careful washing of the cells four times with 2,4-D-free medium and culturing them with 1 µM BL and various concentrations of 2,4-D. Consequently, a considerable improvement was seen in the differentiation rates, with no less than 30% of the cells becoming differentiated into tracheary elements after culturing without 2,4-D (Fig. 2F). To induce tracheary element differentiation, we therefore decided to culture the AC-GT13 cells with 1 µM BL after the removal of 2,4-D. In this differentiation system, cells with ambiguous secondary walls were scarcely observed and were not counted as tracheary elements.
To investigate when and how the tracheary elements were formed, cell sections were stained with toluidine blue and observed under a light microscope. Figure 3A shows the cells 48 h after the start of culture. Almost all the cells were rich in cytoplasm and were equally stained purple. However, the tracheary elements, recognized as blue-stained patterns of the secondary walls, appeared in the cells 72 h after culture (Fig. 3B). In cells 96 h after culture, an assembly of mature tracheary elements had been formed mainly in the inner cell clumps (Fig. 3C), although the tracheary elements had appeared occasionally on the surface of the cell clumps as shown in Figure 2.
The frequencies of tracheary elements at each period, as shown in Figure 3D, suggest that tracheary elements were formed semisynchronously from 48 to 96 h after culture. Finally, more than 30% of the cells had differentiated into tracheary elements after 120 h. We also tried to induce tracheary element differentiation using Col-0 cells, the parental cell line of AC-GT13, and the tracheary elements appeared in the similar time course as AC-GT13 cells, although the lower differentiation rates were obtained (data not shown). There is a possibility that the AC-GT13 cells had been cloned from a single cell that had the higher potential for tracheary element differentiation than other cells in the Col-0 cell suspension.
The AC-GT13 cells were able to stably express GFP-tubulin, through which microtubules could be observed in living cells. In AC-GT13 cells cultured in normal medium, the characteristic microtubule structures representing each stage of the plant cell cycle could be observed, and the same microtubule structures were also observed by immunofluorescent staining with anti-tubulin antibody (Supplemental Fig. 1). This indicated that the AC-GT13 cells were indeed suitable for microtubule observations. If the cells were observed 72 h after the beginning of culture in induction medium, another microtubule structure and secondary wall thickening began to be observed (Fig. 4). To obtain the highest resolution in microscopy, we selected and observed the cells in the most outer layers of the clumps. At first, the randomized cortical microtubules were observed (Fig. 4B, 0 h), and then the microtubules seemed to accumulate as broad bands (Fig. 4B, 2 h). Subsequently, the microtubule bundles appeared (Fig. 4B, 4 h, arrowhead) and the secondary wall thickening began to be seen at the same time and same position as the microtubule bundle (Fig. 4A, 4 h, arrow). Interestingly, at 6 h, the microtubule bundle seemed to be separately arranged into a pair of bundles that located along both sides of the secondary wall (Fig. 4B, 6 h, arrowheads). This should be carefully interpreted because it is possible that the focus of the microscope couldn't reach the bottom of the secondary walls, and high background of cytosolic GFP signals might hide the microtubules just beneath the walls. However, the confocal sections shown in Figure 4C clearly reached the depth under the secondary walls, and microtubules were seldom observed beneath the secondary walls (Fig. 4C, arrowheads). The immunofluorescent microscopy, which had much lower background, also showed the microtubule bundles along both sides of the secondary walls (Fig. 4, D–G). These results demonstrate that the single microtubule bundle certainly split into two bundles, and each of them located along each side of the secondary wall.
Simultaneous Observations of Microtubules and Secondary Walls in Tracheary Element Development
The secondary walls could be stained with fluorescein-conjugated wheat germ agglutinin (WGA) that recognizes hemicelluloses (Hogetsu, 1990
Effects of Microtubule Inhibitors on Secondary Wall Formation
Tracheary elements formed from the AC-GT13 cells possessed various secondary wall patterns (Fig. 6, A–C). In previous studies, colchicine was used widely to investigate the function of microtubules during secondary wall deposition and was known to disorganize the secondary wall patterns (Pickett-Heaps, 1967
Then the inhibitors were added after the secondary walls and microtubule bundles had appeared. In the colchicine-treated cell, the smooth secondary walls became highly disordered by 8 h later, even though the pattern of the secondary wall itself was almost conserved (Fig. 7A). This suggests that the microtubule bundles that associated with both sides of the secondary walls were somehow stabilizing or equalizing secondary wall deposition. In the taxol-treated cell, the cytoplasmic GFP signals were extremely weak, reflecting the effects of taxol (Fig. 7B). To quantify the effects of taxol on microtubule rearrangement, the distances between the pair of microtubule bundles were measured at 2-h intervals at three positions in each of three control and taxol-treated cells. In the taxol-treated cells, the distances were clearly shorter than those in the control cells (Fig. 7C), and their average velocity of microtubule bundle separation was two-thirds that of control cells (Fig. 7D). In the taxol-treated cells, the secondary walls were widening but the growth rate was clearly decreased and resulted in a narrower secondary wall band between the paired microtubule bundles (Fig. 7B). These results suggest that these microtubule bundles are essential not only in the determination of secondary wall patterns but also in the deposition of the secondary walls.
Tracheary Element Differentiation System Using Arabidopsis Cell Suspensions
Although several attempts were previously made to induce tracheary element differentiation, the only practical system available for the study of tracheary element differentiation has been that using zinnia mesophyll cells (Fukuda, 1992
Interestingly, the tracheary elements tended to develop in a congregated manner (Fig. 3C) on the inside of cell clumps. There were several observations that tracheary elements were formed internally as nodules in other suspension cultures (Fukuda, 1992 The advantage of this system is its simple and reproducible culture preparation using repeatedly subcultured suspension cells without regenerating the culture from intact plants. Generally, callus or suspension cells tend to lose their potential for differentiation with time. It is surprising that there seems to be no significant reduction of the ability to reproduce tracheary elements more than 1 year after Col-0 cells were transformed into AC-GT13 cells. This indicates the availability of this system for the model system of tracheary element differentiation in the future.
Several phytohormones are known to participate in tracheary element differentiation (Kuriyama and Fukuda, 2001
Instead of auxin and cytokinin, AC-GT13 cells require brassinosteroid for tracheary element differentiation. Brassinosteroids have been implicated in tracheary element differentiation. Cultured zinnia mesophyll cells synthesize high levels of brassinosteroids for themselves to enter into the final stages of differentiation (Yamamoto et al., 1997
Other phytohormones, such as ethylene and abscisic acid, have also been associated with the tracheary element differentiation process (Kuriyama and Fukuda, 2001
Although the bundling of microtubules associated with secondary wall formation has been observed by transmission electron microscopy or indirect immunofluorescence microscopy in chemically fixed cells, data on the change of microtubule arrangement obtained by these studies are very limited. In this study, therefore, we adopted time-lapse imaging of microtubules in living AC-GT13 cells and showed that microtubules began to accumulate and microtubule bundles appeared before the secondary wall deposition (Fig. 4, A and B, 0 and 2 h). This is the apparent confirmation that microtubules appear at the site where secondary walls will deposit. Subsequently, a single bundle of microtubules under each secondary wall became separately arranged into two microtubule bundles that located along both sides of the nascent secondary wall. They moved apart concomitantly with secondary wall development (Fig. 4, A and B, 4 and 6 h, arrowheads). Such microtubule bundles were also observed by the immunofluorescent microscopy (Fig. 4, E and G). Previously, the microtubules located beside the secondary walls were occasionally observed in cultured zinnia cells (Falconer and Seagull, 1988 The tracheary element differentiation system shown in this study has enabled us not only to observe the microtubules time sequentially but also to investigate the effect of microtubule inhibitors directly. The inhibition of widening of the paired microtubule bundles by taxol (Fig. 7, C and D) suggests that the microtubule bundles are rearranged not by sliding microtubules, but rather via polymerization and depolymerization of tubulin proteins, although the possibility still remains that the microtubule turnover is correlated with the rate of secondary wall deposition and thus taxol itself reduced secondary wall deposition.
In differentiating zinnia mesophyll cells, irregularly patterned secondary walls would form if these microtubules were destroyed (Fukuda and Komamine, 1980b
How do these microtubule bundles regulate secondary wall thickening? In the bordered pits of tracheids, the microtubules were found to accumulate along the edge of the pits in the secondary walls (Hogetsu, 1991 Another possibility is that the secondary walls in AC-GT13 cells may be relatively flat and broad, and the microtubules preferentially accumulate at the sides of the secondary wall ingrowths to regulate active cellulose synthase complexes and/or to deliver vesicles. Previous studies of microtubules and secondary walls in vivo were largely in cylindrical xylem cells. In comparison to them, AC-GT13 cells are somehow spherical, and thus it is expected that such secondary wall development and paired microtubule bundles as observed in this study may be seen in noncylindrical parenchyma cells that are redifferentiating into tracheary elements in vivo. In any case, the 3-D images shown in Figure 5 didn't have enough resolution to demonstrate them. More accurate 3-D reconstruction of secondary wall structures and precise analysis of secondary wall development will resolve this problem.
The sequential development of secondary walls from simple patterns to complex scalariform or reticulate patterns was suggested previously from observations of cultured zinnia mesophyll cells (Falconer and Seagull, 1988
In this study, we have established a system for tracheary element differentiation using the transgenic Arabidopsis cell suspension AC-GT13. Approximately 30% of the cells could semisynchronously differentiate into tracheary elements after 96 h of culture in induction medium. As far as we are aware, this currently is the only system, except for the one in zinnia, available for biochemical or molecular analyses of tracheary element differentiation. We are therefore confident that this system will contribute to the further analysis of processes involved in tracheary element differentiation, especially in secondary wall development. The sequential observation of microtubules in the AC-GT13 cells revealed novel microtubule behavior associated with secondary wall development. The fact that such behavior was not previously recognized, despite the numerous studies conducted on microtubules and secondary walls, clearly demonstrates the superiority of the AC-GT13. Further observations will clarify the processes of microtubule bundling and separation involved in tracheary element differentiation.
Plant Cell Culture
Arabidopsis (Arabidopsis thaliana) Col-0 cells were kindly provided by Dr. Masaaki Umeda. The origin of the Col-0 cells has been described by Mathur et al. (1998)
A 1-mL aliquot of 3-d-old cell suspensions of Arabidopsis Col-0 was added to 3 mL of fresh medium and then inoculated with 100 µL of an overnight culture of Agrobacterium tumefaciens strain LBA4404, transformed with a GFP(S65T)-tubulin alpha (TUA) vector construct (Kumagai et al., 2001
A 1-mL aliquot of 7-d-old AC-GT13 cells was washed four times with induction medium, pH 5.8, containing 4.33 mg L–1 Murashige and Skoog inorganic salts, 170 mg L–1 KH2PO4, 1.0% (w/v) Suc, and 2x B5 vitamins. The cells were then transferred into 10 mL of fresh induction medium, including 1 µM BL in 100-mL culture bottle jars, and then agitated on a rotary shaker at 130 rpm at 23°C in the dark.
The suspension cells were fixed every 24 h after the beginning of induction in a mixture of 45% ethanol, 2.5% acetic acid, and 2.5% (v/v) formalin and rinsed twice with 0.1 M phosphate-buffered saline (PBS; pH 7.0). The cells were dehydrated in a graded series of ethanol (50, 60, 70, 80, 90, 99.5, and 100% [v/v] for 20 min for each step) and embedded in technovit 7100 resin (Kulzer & Co., Wehrheim, Germany). The 0.4-µm-thick sections, cut with an ultramicrotome (Ultracut UCT; Leica Microsystems, Wetzlar, Germany), were stained for 1 min with 0.1% (w/v) Toluidine Blue O (Waldeck GmbH & Co., Munster, Germany) solution in 100 mM PBS, pH 7.0, rinsed with distilled water, and then observed under a light microscope (BX51; Olympus, Tokyo). For measurement of differentiation rates, three sections were independently cut from one sample, and more than 500 cells were examined in each section.
For immunostaining, cells were fixed with 3.7% (v/v) formaldehyde in 50 mM PIPES, 1 mM MgSO4, 5 mM EGTA, and 1% (v/v) glycerol, pH 6.8, for 30 min at room temperature and then treated with an enzyme solution containing 0.5% (w/v) pectolyase and Y23 cellulase Y-C (both from Seishin, Tokyo) in 0.4 M mannitol, 50 mM PIPES, 1 mM MgSO4, 5 mM EGTA, 0.5 mM phenylmethylsulfonyl fluoride, and 1 mg L–1 leupeptin. Then cells were attached to polyethylenimine-coated coverslips. The cells were incubated in 0.1 M Gly, 1% (w/v) bovine serum albumin, and 0.05% (v/v) Triton X-100 in PBS (20 mM sodium phosphate and 150 mM NaCl, pH 7.0) for 15 min and incubated for 1 h with the primary antibody mouse anti-chicken
For the time-sequence observations, 72-h-old AC-GT13 cells, cultured in induction medium with 1 µM BL, were transferred onto 35-mm petri dishes with 27-mm coverslip windows at the bottom (Matsunami Glass Industries, Osaka). The dishes were placed onto the inverted platform of a fluorescence microscope (IX; Olympus), equipped with an UPlanApo 100x/1.35 oil Iris objective lens and a confocal laser scanning head and control systems (CLSM GB-200; Olympus). This observation system was found to be useful for the observation of microtubules in AC-GT13 cells with developing secondary walls. Moreover, by inoculation of the cells with 20 mg L–1 Texas Red-conjugated WGA (Molecular Probes; 2 mg mL–1 stock solution in distilled water) for 30 min before the observations, the microtubules and secondary walls could be followed simultaneously. GFP was excited by argon laser (wavelength 488 nm), Texas Red and rhodamine were excited by helium neon laser (wavelength 543 nm), and each scan of single-confocal sections took 10 s by a confocal laser scanning microscopy (CLSM) system. The confocal sections were reconstructed by maximum-intensity projection using Metamorph (Universal Imaging, Downingtown, Panama) and processed digitally using Photoshop software (Adobe Systems, Mountain View, CA). For the analysis of the 3-D structures of the secondary walls, images were reconstructed using Amira (Indeed-Visual Concepts GmbH, Berlin). For the observations of immunostained cells, a fluorescence microscope (IX; Olympus) equipped with a cooled CCD camera head system (Cool-SNAP HQ, PhotoMetrics, Huntington Beach, Canada) were also used in addition to the CLSM system described above.
After 72 h of culture in induction medium, the AC-GT13 cells were treated with 2 µM taxol (Sigma, St. Louis) or 100 µM colchicine (Sigma), following staining of the secondary walls with WGA, and were then observed 30 or 60 min later by CLSM.
We thank Dr. Csaba Koncz (Max-Planck-Institut für Züchtungsforschung) and Dr. Masaaki Umeda (The University of Tokyo) for providing the Arabidopsis Col-0 cell suspension, and Dr. Hiroo Fukuda (The University of Tokyo) for valuable suggestions. We also thank Arata Yoneda (The University of Tokyo) for technical advice. Received September 7, 2004; returned for revision November 1, 2004; accepted December 28, 2004.
1 This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas from The Ministry of Education, Culture, Sports, Science and Technology, Japan (grant no. 15031209 to S.H.). 
[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.104.052613. * Corresponding author; e-mail hasezawa{at}k.u-tokyo.ac.jp; fax 81–4–7136–3706.
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-tubulin (Oncogene, Darmstadt, Germany) in PBS. The specimens were then washed three times in PBS and incubated for 1 h with the secondary antibody rhodamine-conjugated goat anti-mouse antibody (Cappel, West Chester, PA). Then cells were washed three times with PBS and embedded in a glycerol solution containing an antifading reagent (SlowFade Light; Molecular Probes, Eugene, OR). 
