Plant and Cell Physiology Advance Access originally published online on September 17, 2007
Plant and Cell Physiology 2007 48(10):1393-1403; doi:10.1093/pcp/pcm120
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Rapid Paper |
Chemical Genetic Screening Identifies a Novel Inhibitor of Parallel Alignment of Cortical Microtubules and Cellulose Microfibrils
1Plant Science Center, RIKEN, 1-7-22, Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045 Japan
2Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwanoha 5-1-5, Kashiwa, Chiba, 277-8562 Japan
3Discovery Research Institute, RIKEN, 2-1, Hirosawa, Wako, Saitama, 351-0198 Japan
*Corresponding author: E-mail, minami{at}postman.riken.go.jp; Fax, +81-45-503-9586.
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Abstract |
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It is a well-known hypothesis that cortical microtubules control the direction of cellulose microfibril deposition, and that the parallel cellulose microfibrils determine anisotropic cell expansion and plant cell morphogenesis. However, the molecular mechanism by which cortical microtubules regulate the orientation of cellulose microfibrils is still unclear. To investigate this mechanism, chemical genetic screening was performed. From this screening, ‘SS compounds’ were identified that induced a spherical swelling phenotype in tobacco BY-2 cells. The SS compounds could be categorized into three classes: those that disrupted the cortical microtubules; those that reduced cellulose microfibril content; and thirdly those that had neither of these effects. In the last class, a chemical designated ‘cobtorin’ was found to induce the spherical swelling phenotype at the lowest concentration, suggesting strong binding activity to the putative target. Examining cellulose microfibril regeneration using taxol-treated protoplasts revealed that the cobtorin compound perturbed the parallel alignment of pre-existing cortical microtubules and nascent cellulose microfibrils. Thus, cobtorin could be a novel inhibitor and an attractive tool for further investigation of the mechanism that enables cortical microtubules to guide the parallel deposition of cellulose microfibrils.
Keywords: Cell swelling - Cellulose microfibril - Chemical genetics - Cortical microtubule - Inhibitor
Abbreviations: APM, amiprofos methyl; DCB, 2,6-dichlorobenzonitrile; GFP, green fluorescent protein
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgmentsReferences |
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Plant cells are surrounded by rigid cell walls that prevent them from changing their shape or position easily. Because the wall restricts the direction of cell expansion or elongation, its formation is an important factor in plant cell morphogenesis. It is considered that cellulose microfibrils are the main mechanical element of the cell wall, and it is generally considered that cortical microtubules control the direction of cellulose microfibril deposition (Baskin 2001
, Baskin 2005). If cortical microtubule organization (Upadhyaya and Noodén 1978, Whittington et al. 2001, Baskin et al. 2004) or cellulose microfibril deposition (Sugimoto et al. 2001, Williamson et al. 2001) are inhibited by drugs or genetic mutation, plant cells lose their anisotropy and show swelling. The parallel alignment of cortical microtubules and cellulose microfibrils was confirmed by transmission electron microscopy and immunofluorescence microscopy using fixed cells (Giddings and Staehelin 1991, Hasezawa and Nozaki 1999), and more recently, by visualization of cellulose synthase and cortical microtubules in living cells using yellow fluorescent protein-labeled cellulose synthase (YFP:CESA6) and cyan fluorescent protein-labeled 
-tubulin (CFP:TUA1) (Paredez et al. 2006). From these observations, it became clear that cellulose synthase complexes move along linear paths coincident with cortical microtubules; however, it still remains unclear how cortical microtubules guide the movement of the cellulose synthase complex.
Despite numerous studies using recent genetic techniques, including the generation of loss-of-function mutants and gain-of-function activation lines with altered plant and cell morphology, few reports have shown loss of the relationship between cortical microtubules and cellulose microfibrils. One protein of interest is COBRA (COB), an extracellular glycosylphosphatidylinositol (GPI)-anchored protein, which is expressed during rapid cell expansion. The cells in the root elongation zone of cob mutants exhibit severe radial swelling and defects in longitudinal elongation (Schindelman et al. 2001, Roudier et al. 2005). Interestingly, cob mutants occasionally have disordered cellulose microfibrils (Roudier et al. 2005), although the orientation of cortical microtubules is not significantly affected in the root elongation zone (Hauser et al. 1995). Another interesting protein is MICROTUBULE ORGANIZATION 1 (MOR1), a plant microtubule-associated protein in the MAP215 family, which is essential for cortical microtubule organization. It has been reported that mor1 mutants have fragmented cortical microtubules and lose cell anisotropy (Whittington et al. 2001), although the cellulose microfibrils were not reduced and did not alter their alignment (Sugimoto et al. 2003, Wasteneys and Fujita 2006). Although COBRA and MOR1 are interesting genes with which to investigate plant cell morphogenesis, new genetic or chemical tools may be required to reveal the putative linkage mechanism between the cortical microtubules and the cellulose synthase complexes.
Unfortunately, conventional genetic analysis has not yet revealed this mechanism. Several difficulties are encountered in genetic screens: for example, a single mutation may not cause a detectable phenotype because of gene redundancy, a mutation in a particular gene may be lethal, or a mutation may cause unexpected phenotypes because the defective protein interacts with several other proteins and/or participates in many functions. In such cases, ‘chemical genetics’ has several advantages: (i) chemicals may dominantly inhibit the function of the target; (ii) the compounds may attack proteins with similar structures and functions, thus overcoming the problems of redundancy; (iii) inhibition is inducible at any time during the experiment or at any stage of cell development; and (iv) the degree of the effect may be controlled through varying the dose of the chemical compounds.
In this study, we tested the chemical genetics approach, screening two chemical libraries for compounds that affected the cortical microtubule–cellulose microfibril-dependent cell morphogenesis pathway. As it was expected that inhibition of the pathway would cause cell swelling, we screened for a spherical swelling phenotype using the tobacco BY-2 cell line. Among the 19 ‘SS compounds’ that induced spherical swelling, the SS17 compound, which we designated ‘cobtorin’, neither disrupted cortical microtubules nor reduced cellulose microfibril content, but instead perturbed the parallel alignment of pre-existing cortical microtubules and nascent cellulose microfibrils. The data in this report suggest that the target of cobtorin may have an important role in the relationship between cortical microtubules and the cellulose synthase complex. Thus, cobtorin could be a novel and attractive tool with which to investigate further the mechanism that enables cortical microtubules to guide the cellulose synthase complex movement along them, resulting in the parallel alignment of cortical microtubules and cellulose microfibrils.
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Results |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgmentsReferences |
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Identification of spherical swelling phenotype
To screen for new inhibitors that affect cell morphogenesis, we used two chemical libraries, LATCA and Spectrum, which together contained a total of approximately 4,000 small bioactive compounds (see Materials and Methods). Cells of the tobacco suspension line BY-GT, which stably express a green fluorescent protein (GFP)–
-tubulin fusion protein, allowing visualization of microtubules in living cells (Kumagai et al. 2001), were cultured for 2 d in a medium containing 25 µM of each chemical compound in a 96-well plate, and then screened for changes in cell morphology. Various phenotypes were seen including cell death, long or short cells, cytoplasmic strand-less cells, cells with aberrant division planes and notably spherically swollen cells (Fig. 1). While normal BY-GT cells have a cylindrical shape, and continue unidirectional elongation and transverse division (Fig. 1A), cells with the spherical swelling phenotype showed aberrant expansion: partial cell swelling in a weak phenotype (Fig. 1B, arrowheads), and a wholly globular shape in a strong phenotype (Fig. 1C). In some cases, protoplast-like cells that had almost lost cell–cell adhesion were also observed (data not shown).
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After a comprehensive first screening followed by a second screen for reproducibility and dose dependence, 21 compounds (18 from LATCA and three from Spectrum) were selected as agents responsible for the spherical swelling phenotype. We named these candidates ‘SS compounds’ (SS01–21). As we speculated that such spherical swelling phenotypes could be caused by cell wall insufficiency, two known drugs were tested for comparison: amiprofos methyl (APM), a microtubule polymerization inhibitor, and 2,6-dichlorobenzonitrile (DCB), a cellulose biosynthesis inhibitor. Both APM- and DCB-treated cells exhibited a typical spherical swelling phenotype, which was similar to that seen in our screen (Fig. 1D, E).
Classification of SS compounds by the effect on cortical microtubules and cellulose microfibrils
To classify the mode of action of the SS compounds, their effects on cortical microtubule arrays and cellulose microfibril deposition were analyzed. The cortical microtubule array was observed using confocal laser scanning microscopy at 4 h after the application of the chemical candidates, when the cells had not yet changed their shape to the spherical form. Distinct fibrous cortical microtubules were observed as a mainly transverse array in the control BY-GT cells (Fig. 2A). When cells were treated with APM, no fibrous structures could be found and only dim fluorescence in the cytoplasm was observed (Fig. 2B). In contrast, DCB-treated cells had an obvious cortical microtubule array similar to the control (Fig. 2C), and there seemed to be no effect on microtubules. Addition of SS compounds to the cells resulted in one of three classes of effect on cortical microtubules: (+) no significant effect (Fig. 2D); (±) cortical microtubules were partially disrupted, with a few transverse microtubules remaining (Fig. 2E); and (–) cortical microtubules completely disappeared (Fig. 2F). We did not observe directional changes, bundling or short fragmentation of cortical microtubules.
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To estimate and classify cellulose deposition in the cell wall, cells were stained with calcofluor and observed by fluorescence microscopy, and then fluorescent intensities were measured. While control cells showed strong calcofluor fluorescence, those of DCB-treated cells seemed to be much weaker (Fig. 3A). Intensity profiles were obtained for the lines shown in Fig. 3A (profiles displayed in Fig. 3B), and the average scores of the maximum intensity from >30 cells were assembled (Fig. 3C). DCB-treated cells had <50% the calcofluor fluorescence of the control, while APM-treated cells showed almost the same fluorescence. Fluorescent intensities of calcofluor were then measured in cells treated with the SS compounds (Fig. 3D). We observed roughly three categories: (+) approximately the same fluorescent intensity as the control; (±) 20–30% decrease; and (–) >50% loss.
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The data described above along with the names of the SS compounds are summarized in Table 1. We found that two pairs of the 21 SS compounds were identical, so the final number of independent compounds identified was 19. In the dose dependency test, a plus (+) score was assigned to chemically treated cells exhibiting strong spherical swelling as shown in Fig. 1C, and plus/minus (±) indicates the weak phenotype as in Fig. 1B. Most of the SS compounds caused a spherical swelling phenotype at 25 and 5 µM, and some could also induce morphological changes at lower concentrations. The CMT column in Table 1 shows the results of cortical microtubule observations (Fig. 2), and the CMF column shows a summary of the cellulose contents estimated by calcofluor fluorescence (Fig. 3D). From these data, we classified the SS compounds into three groups: class I were those that caused cortical microtubule disorganization; class II compounds had no visible effects on either cortical microtubules or cellulose microfibrils; and class III compounds caused a reduction in cellulose microfibril content. Class I included some known anti-microtubule agents, and class III contained dichlobenil, which is a synonym of DCB.
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The structures of the SS compounds are shown in Fig. 4. No significant structural similarity was found among the compounds.
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Relationship between cortical microtubule orientation and cellulose microfibril deposition
Of the SS compounds, SS17, 4-[(2-chlorophenyl)methoxy]-1-nitrobenzene, had the highest activity, since the spherical swelling phenotype could be strongly induced in BY-GT cells at a final concentration of <200 nM (Table 1). BY-2 cells incubated with 1 µM SS17 showed spherical swelling (Fig. 5A) and Arabidopsis hypocotyl cells grown in the dark with 1 µM SS17 exhibited severely short and swollen cells compared with the longitudinal cells of hypocotyls grown in control medium (Fig. 5C). We therefore designated SS17 ‘cobtorin’ after the Japanese word ‘cobutori’, indicating the appearance of a plump person. To analyze the mode of action of cobtorin, we first confirmed its effect on cell morphogenesis and microtubules. At 4 h after application of cobtorin, cells had distinct transverse cortical microtubules (not shown, classified as CMT + in Table 1). After 2 d incubation with 1 µM cobtorin, cells had the spherical swelling phenotype (Fig. 5A) with parallel cortical microtubules, similar to the control cells (Fig. 5B). Furthermore, we observed cell shape and cortical microtubule orientation in etiolated Arabidopsis hypocotyls which were grown on agar medium with or without cobtorin for a week. The hypocotyl cells exhibited severe morphological changes under 1 µM cobtorin (Fig. 5C), but distinct cortical microtubules were still observed in the transverse direction at 7 d after germination (Fig. 5D). This suggested that cobtorin indeed induced the cell spherical swelling phenotype without any cortical microtubule depolymerization or disorganization.
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We next performed a BY-2 synchronization assay with or without 1 µM cobtorin. After 24 h incubation with aphidicolin, an inhibitor of DNA polymerase
, the cell cycle is arrested at G1/S phase. Upon release from aphidicolin, cells re-enter the cell cycle simultaneously. The cells were stained with 4',6-diamidino-2-phenylindole (DAPI), and the mitotic index (the ratio of cells in mitosis to total cells) was calculated for each treatment. We found that the peak time and the maximum value of the mitotic index in the cobtorin-treated cells were nearly identical to those in the control cells (data not shown). This might indicate that in addition to cortical microtubules, cell cycle-regulated microtubule arrays (pre-prophase band, spindle and phragmoplast) are likewise unaffected by cobtorin.
We also examined cellulose microfibril regeneration in cobtorin-treated protoplasts that had been pre-treated with the microtubule-stabilizing agent taxol (Hasezawa and Nozaki 1999). Protoplasts are suitable for observing nascent cellulose microfibril regeneration because the pre-existing cell wall has been digested and only newly synthesized cellulose microfibrils are detected by calcofluor staining. However, protoplast preparation usually results in disorganization of cortical microtubules to a rather random array (Hasezawa et al. 1988), and furthermore there seems to be no relationship between the direction of cortical microtubules and cellulose microfibrils even in control cells (data not shown, Hasezawa and Nozaki 1999). Therefore, to preserve the parallel cortical microtubule arrays in BY-2 cells, we used protoplasts derived from taxol-treated cells. We concluded that taxol treatment did not affect cellulose synthesis, because nascent cellulose microfibrils could be observed during the same time course as in non-treated protoplasts (data not shown). The BY-2 protoplasts were incubated with taxol, stained with calcofluor and then observed by confocal laser scanning microscopy. At 1–1.5 h after preparation, the regenerated cellulose microfibrils were seen as fibrous structures on the cortex of the protoplasts. In the control cells, the alignment of the cortical microtubules visualized by GFP– -tubulin fusion protein and the cellulose microfibrils stained by calcofluor seemed to be very similar and in parallel orientation (Fig. 6A, control). On the other hand, when the protoplasts were cultured with 1 µM cobtorin, the cellulose microfibrils seemed to be deposited in quite different directions from the cortical microtubules (Fig. 6A, cobtorin). We quantified orientation by calculating the average angles of the fibers against the horizon (see Materials and Methods and Supplementary Fig. S1). The average angle of microtubules in the control cell was 55.5°, whereas that of cellulose microfibrils was very similar (56.2°) (Fig. 6B), indicating highly parallel orientations. On the other hand, the average angles of microtubules and cellulose microfibrils in the cobtorin-treated cells were 38.7° and 107.7°, respectively (Fig. 6B), indicating that they were oriented in quite different directions. The values of the difference in the angle between the microtubules and the cellulose microfibrils were plotted for each of 24 cells (Fig. 6C). Differences in the control cells were small, whereas those under cobtorin were highly dispersed. The averages of the difference in control and cobtorin were 9.7 ± 1.4° and 32.8 ± 5.0°, respectively, and there was a highly significant difference between them (P < 0.0005, Mann–Whitney's U-test). These data strongly indicated that cobtorin inhibited the parallel alignment of cortical microtubules and nascent cellulose microfibril deposition.
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgmentsReferences |
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Chemical genetics screening
Genetic and genomic approaches have been very widely used to study plant biological systems. In forward genetics, random mutations are induced in the plant genome, followed by phenotypic screening and gene identification to reveal the function of genes. Chemical genetics is an alternative technique to study biological systems that uses small molecules instead of genetic manipulations (Spring 2005
). In ‘forward’ chemical genetics, a large number of chemical compounds are used to screen for an expected phenotype, and then the mode of action of any candidate molecules is investigated.
The use of an efficient library is important for high-throughput screening (Irwin 2006). We used two chemical libraries in this study, Spectrum and LATCA. Spectrum is a commercial library containing 2,000 ‘biologically active and structurally diverse’ compounds (Microsource Discovery Systems). The LATCA (‘library of active compounds on Arabidopsis’) library consists of 2,080 small molecules from three commercial libraries that have been pre-screened for compounds that inhibit growth of etiolated Arabidopsis hypocotyls by >30% (Chow et al. 2006). In this study, a larger number of chemical candidates causing spherical swelling of cells was found in the LATCA than in the Spectrum library. Therefore, the pre-screened LATCA library seemed highly efficient for morphological screening in plant cells. However, it might be expected that screening using the LATCA library would present difficulties in identifying suppressive mechanisms, because inhibition of the suppressor would result in a promotional phenotype. In such a case, the non-biased Spectrum library may have advantages.
Chemical genetic screening is often rapid and easy; there is no need for mutagenesis and maintenance of mutation lines, and the chemicals can be applied at any time point during experiments or at any developmental stage. We used a tobacco BY-2 suspension cell line, because it grows very quickly, has a uniform cell structure and is suitable for cell morphological observations. Therefore, the combination of BY-2 and a chemical genetics approach enabled cell morphological screening at the single cell level to select compounds that affect the mechanisms of plant cell morphogenesis.
Mode of action of SS compounds
We screened the 19 SS compounds twice and categorized them into three classes at a third screening by observation of cortical microtubule orientation and cellulose microfibril fluorescent intensity. In class I, which disrupted cortical microtubules, several known drugs were identified: APM and pronamide (synonym of propyzamide) are well-known anti-microtubule agents, and terbutol (Lehnen Jr. et al. 1990) and trifluralin (Hess and Bayer 1977, Anthony and Hussey 1999, Nyporko et al. 2002) have also been reported to affect the microtubules or the microtubule-organizing center. Many anti-microtubule drugs have been used as herbicides, and it has been reported that use of microtubule-depolymerizing agents causes cell or tissue swelling (Lignowski and Scott 1971, Upadhyaya and Noodén 1978, Katsuta and Shibaoka 1988, Anthony and Hussey 1999, Baskin et al. 2004). Identification of these known chemical compounds during the screen and their categorization in class I provide a validation of our methods.
In this context, it is also appropriate that dichlobenil (DCB) was assigned to class III (decreased cellulose microfibril content) because DCB is known as a cellulose biosynthesis inhibitor. The fluorescent intensity of calcofluor may not strictly reflect the accurate cellulose microfibril content; however, it is useful for classification. Other candidates in class III are novel compounds, but SS14 has the same substructure as morlin (Debolt et al. 2007) or coumarin (Goodwin and Taves 1950, Harada et al. 1972, Hara et al. 1973). These are both reported to cause tissue or cell swelling and cellulose synthesis reduction, confirming that SS14 could have a similar ability to induce the spherical swelling phenotype and a decrease in cellulose microfibril content. In contrast, SS14 did not affect cortical microtubule orientation at all, although morlin inhibited microtubule dynamics as well as causing cortical microtubule fragmentation and bundling (Debolt et al. 2007). Other SS compounds in class III might inhibit cellulose synthesis directly or indirectly by affecting substrate synthesis or transport.
Class II contained a known compound, dihydro-obliquin, but we only found from the literature that it is an artificially hydrogenated obliquin derivative (Dean and Parton 1969). Obliquin was isolated as a phenylated coumarin from a timber tree, Ptæroxylon obliquum (Dean and Taylor 1966, Koorbanally et al. 2002), but we could not find any reports about its biological activity or that of dihydro-obliquin. Therefore, the mode of action of the SS compounds in class II could not be predicted from the literature.
A novel inhibitor of cortical microtubule–cellulose microfibril parallel alignment
We were most interested in the class II SS compounds, because they affected neither cortical microtubule orientation nor cellulose microfibril synthesis but nevertheless caused the spherical swelling phenotype. It is a well-known hypothesis that cortical microtubules regulate the cellulose microfibril deposition pattern, and alignment of cellulose microfibrils restricts the direction of cell anisotropic growth (Giddings and Staehelin 1991, Baskin 2001, Wasteneys 2004). The class I and class III compounds may inhibit cortical microtubule organization and cellulose microfibril synthesis, respectively, but class II compounds did not seem to affect either. Therefore, it is possible that they disrupt the directional relationship between cortical microtubule orientation and cellulose microfibril deposition.
Among the class II compounds, cobtorin had the highest bioactivity, probably indicating that the induction of the spherical swelling phenotype is not a side effect but a specific phenomenon, and that cobtorin may have strong binding affinity for a putative target protein. It would be a benefit during further investigation to identify the target, for example by affinity purification or resistant mutant screening. To analyze the mode of action of cobtorin, we examined cellulose microfibril regeneration using taxol-treated protoplasts (Hasezawa and Nozaki 1999). The protoplasts without cobtorin regenerated cellulose microfibrils parallel to cortical microtubules; however, the protoplasts with cobtorin exhibited quite different orientations of the microtubules and microfibrils. This result indicated that cobtorin disrupted the parallel alignment of the pre-existing cortical microtubule array and nascent cellulose microfibril deposition, perhaps attacking the putative linkage mechanism between the cortical microtubules and cellulose synthase complexes. To reveal the inhibitory mechanism of cobtorin, identification of its target may be required. Cobtorin could be a novel and attractive tool to use for investigating the regulatory mechanism by which cortical microtubules influence the cellulose microfibril deposition pattern.
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Materials and Methods |
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Plant material and growth conditions
Suspension cultures of a BY-GT (BY-2 cells expressing GFP–
-tubulin fusion protein) cell line (Kumagai et al. 2001) were maintained similar to the tobacco BY-2 cell line (Nagata et al. 1992). Briefly, at weekly intervals, suspension cultures of the BY-GT cell line were diluted 80-fold with a modified Linsmaier and Skoog medium (Kumagai et al. 2001). The cell suspension was agitated on a rotary shaker at 130 r.p.m. at 27°C in the dark.
The transgenic Arabidopsis line expressing GFP– -tubulin was in the Columbia ecotype (Ueda et al. 1999). Seedlings were germinated and grown in the dark at 22°C on plates containing Murashige and Skoog plant salt mixture (Wako Pure Chemical Industries, Ltd, Osaka, Japan), Gamborg vitamin mixture (Sigma Chemical Co., St Louis, MO, USA), 0.05% MES and 0.8% agar.
Chemical libraries and screens
Two 96-well format chemical libraries, Spectrum and LATCA, were screened. Spectrum is a commercial library from Microsource (Microsource Discovery Systems, Inc., CT, USA), containing 2,000 biologically active and structurally diverse compounds. The LATCA library consists of 2,080 small molecules that have been found to inhibit growth of etiolated Arabidopsis hypocotyls (Chow et al. 2006). This library was assembled from three commercial libraries: Diverset (Chembridge, CA, USA), LOPAC (Sigma-Aldrich, MA, USA) and Spectrum (Microsource). All compounds are dissolved in dimethylsulfoxide (DMSO) at 2.5 or 5 mM each, and stored at –80°C.
For the first screening, each chemical compound was added to 60 µl of 2-day-old BY-GT cell culture in a 96-well plate at a final concentration of 25 µM, and the plates were incubated at 27°C in the dark for 2 d. Plates were placed onto the inverted platform of a fluorescence microscope (IX81, Olympus Co. Ltd, Tokyo, Japan) equipped with a cooled CCD camera head system (DP70, Olympus). Each well was semi-automatically photographed using LuminaVision software (Mitani-corp., Tokyo, Japan), and then each image was observed and scored to classify the cell morphology. In the second screening, each candidate identified from the first screening was diluted with DMSO, added to cell culture at a final concentration of 25 µM, 5 µM, 1 µM, 200 nM and 40 nM, and then analyzed with the same procedures as before. Data from screened compounds were managed and analyzed using Instant JChem software (ChemAxon, Budapest, Hungary) and ChemMine (http://bioweb.ucr.edu/ChemMineV2/).
Cell staining and observation
To analyze microtubule alignment, BY-GT cells were observed at 4 h after application of the chemical compounds at a final concentration of 25 µM using an inverted fluorescence microscope (AxioVert 200M, Zeiss, Oberkochen, Germany) equipped with a confocal laser scanning head system (LSM 510, Zeiss). Subsequently, images were digitally processed using Photoshop software (Adobe Systems Inc., CA, USA).
To stain cellulose microfibrils, BY-GT cells were incubated with calcofluor (Fluorescent Brightener 28, MP Biomedicals Inc., Illkirch, France) at a final concentration of 0.01% (w/v) for 15 min. After incubation, the cells were photographed using fluorescence microscopy as described above. The intensity profiles of the fluorescence were obtained using ImageJ software (Abramoff et al. 2004, National Institutes of Health, MD, USA, http://rsb.info.nih.gov/ij/).
Protoplast isolation
For preparing protoplasts, 4-day-old BY-GT cells were treated with 20 µM taxol (Sigma) for 1.5 h at 27°C, and then with an enzyme solution containing 1% Cellulase ‘ONOZUKA’ RS (Yakult Honsha Co., Ltd, Tokyo, Japan) and 0.1% Pectolyase Y-23 (Kyowa Chemical Products Co., Ltd, Osaka, Japan) in 0.35 M mannitol and 20 µM taxol for 1.5 h at 30°C to digest the cell wall completely (Hasezawa and Nozaki 1999). Subsequently the isolated protoplasts were washed twice with 0.4 M mannitol and 20 µM taxol, and were resuspended in modified Linsmaier and Skoog medium with 0.32 M mannitol, 1% sucrose, 0.1 mg l–1 -naphthalene acetic acid, 1 mg l–1 benzylaminopurine and 20 µM taxol for 1.5 h at 27°C to regenerate cell walls. The cobtorin compound was added at a final concentration of 1 µM after the enzymes were washed out. The protoplasts were transferred into 
35 mm Petri dishes with poly-L-lysine-coated 14 mm coverslip windows at the bottom (Matsunami Glass Ind., Ltd, Osaka, Japan). The protoplasts on the dishes were photographed using confocal laser scanning microscopy as described above.
Image analysis
In order to estimate the angles of cortical microtubules or cellulose microfibrils, we performed a semi-automated image angle measurement (Supplementary Fig. S1). We firstly obtained skeletonized images (Supplementary Fig. S1D) via skeletal enhancement by a 9–12 pixel-band-pass filter (Supplementary Fig. S1A, B) and binarization by intensity thresholding (Supplementary Fig. S1B, C) from confocal images of microtubules or cellulose microfibrils labeled with GFP– -tubulin or calcofluor, respectively. In the skeletonized images, angles of a pixel pair could be categorized into 0, 45, 90 and 135° against the horizon, and their pixel pair numbers were counted (Supplementary Fig. S1E, F). Mean angle 
is defined by
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where n0, n45, n90 and n135 are the numbers of the pixel pair, which form the 0, 45, 90 and 135°, respectively. All procedures were semi-automatically performed using our Java plug-ins; BandPass, BilevelThin and LineAngle (http://www.biol.s.u-tokyo.ac.jp/users/hasezawa/kbi/ij_plugins/index.html) on ImageJ.
Supplementary material
Supplementary material mentioned in the article is available to online subscribers at the journal website www.pcp.oxfordjournals.org.
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Acknowledgments |
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We are grateful to Dr. S. Cutler (University of California) for the kind gift of the LATCA chemical library, and to Dr. T. Hashimoto (Nara Institute of Science and Technology) for the kind gift of the Arabidopsis GFP–
-tubulin line. This work was supported by a Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists to A.Y. |
References |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgmentsReferences |
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Abramoff MD, Magelhaes PJ, Ram SJ. Image processing with ImageJ. Biophoton. Int. (2004) 11:36–42.
Anthony RG, Hussey PJ. Dinitroaniline resistance and the microtubule cytoskeleton. Trends Plant Sci. (1999) 4:112–116.[CrossRef][ISI][Medline]
Baskin TI. On the alignment of cellulose microfibrils by cortical microtubules: a review and a model. Protoplasma (2001) 215:150–171.[CrossRef][ISI][Medline]
Baskin TI. Anisotropic expansion of the plant cell wall. Annu. Rev. Cell Dev. Biol. (2005) 21:203–222.[CrossRef][ISI][Medline]
Baskin TI, Beemster GT, Judy-March JE, Marga F. Disorganization of cortical microtubules stimulates tangential expansion and reduces the uniformity of cellulose microfibril alignment among cells in the root of Arabidopsis. Plant Physiol. (2004) 135:2279–2290.
Chow F, Alfred S, Zhao Y, Fung P, Cutler S. LATCA: a library of biologically active small molecules for plant chemical genomics. The 17th International Conference of Arabidopsis Research. (2006) 11.
Dean FM, Parton B. The structure and synthesis of (t)-obliquin. J. Chem. Soc. (1969) C:526–531.
Dean FM, Taylor D.AH. Extractives from East African timbers. part II. Ptaroxylon obliquum. J. Chem. Soc. (1966) C:114–116.
Debolt S, Gutierrez R, Ehrhardt DW, Melo CV, Ross L, Cutler SR, Somerville C, Bonetta D. Morlin, an inhibitor of cortical microtubule dynamics and cellulose synthase movement. Proc. Natl Acad. Sci. USA (2007) 104:5854–5859.
Giddings TH, Staehelin LA. Microtubule-mediated control of microfibril deposition: a re-examination of the hypothesis. In: The Cytoskeletal Basis of Plant Growth and Form—Lloyd CM, ed. (1991) London: Academic Press. 85–99.
Goodwin RH, Taves C. The effect of coumarin derivatives on the growth of Avena roots. Amer. J. Bot. (1950) 37:224–231.[Medline]
Hara M, Umetsu N, Miyamoto C, Tamari K. Inhibition of the biosynthesis of plant cell wall materials, especially cellulose biosynthesis, by coumarin. Plant Cell Physiol. (1973) 14:11–28.
Harada H, Oyama K, Chervel J. Effect of coumarin and other factors on the modification of form and growth of isolated mesophyll cells. Z. Pflanzenphysiol. (1972) 66:307–324.
Hasezawa S, Hogetsu T, Syono K. Rearrangement of cortical microtubules in elongating cells derived from tobacco protoplasts—a time-course observation by immunofluorescence microscopy. J. Plant Physiol. (1988) 133:46–51.[ISI]
Hasezawa S, Nozaki H. Role of cortical microtubules in the orientation of cellulose microfibril deposition in higher-plant cells. Protoplasma (1999) 209:98–104.[CrossRef][ISI]
Hauser MT, Morikami A, Benfey PN. Conditional root expansion mutants of Arabidopsis. Development (1995) 121:1237–1252.[Abstract]
Hess FD, Bayer DE. Binding of the herbicide trifluralin to Chlamydomonas flagellar tubulin. J. Cell Sci. (1977) 24:351–360.[Abstract]
Irwin JJ. How good is your screening library? Curr. Opin. Chem. Biol. (2006) 10:352–356.[CrossRef][ISI][Medline]
Katsuta J, Shibaoka H. The roles of the cytoskeleton and the cell wall in nuclear positioning in tobacco BY-2 cells. Plant Cell Physiol. (1988) 29:403–413.
Koorbanally NA, Randrianarivelojosia M, Mulholland DA, Quarles van Ufford L, van den Berg AJ. Bioactive constituents of Cedrelopsis microfoliata. J. Nat. Prod. (2002) 65:1349–1352.[CrossRef][Medline]
Kumagai F, Yoneda A, Tomida T, Sano T, Nagata T, Hasezawa S. Fate of nascent microtubules organized at the M/G11 interface, as visualized by synchronized tobacco BY-2 cells stably expressing GFP-tubulin: time-sequence observations of the reorganization of cortical microtubules in living plant cells. Plant Cell Physiol. (2001) 42:723–732.
Lehnen L.P. Jr, Vaughan MA, Vaughn KC. Terbutol affects spindle microtubule organizing centres. J. Exp. Bot. (1990) 41:537–546.
Lignowski EM, Scott EG. Trifluralin and root growth. Plant Cell Physiol. (1971) 12:701–708.
Nagata T, Nemoto Y, Hasezawa S. Tobacco BY-2 cell line as the ‘Hela’ cell in the cell biology of higher plants. Int. Rev. Cytol. (1992) 132:1–30.[ISI]
Nyporko AY, Yemets AI, Klimkina LA, Blume YB. Sensitivity of Eleusine indica callus to trifluralin and amiprophosmethyl in correlation with the binding of these compounds to tubulin. Russian J. Plant Physiol. (2002) 49:413–418.[CrossRef][ISI]
Paredez AR, Somerville CR, Ehrhardt DW. Visualization of cellulose synthase demonstrates functional association with microtubules. Science (2006) 312:1491–1495.
Roudier F, Fernandez AG, Fujita M, Himmelspach R, Borner GH, et al. COBRA, an Arabidopsis extracellular glycosyl-phosphatidyl inositol-anchored protein, specifically controls highly anisotropic expansion through its involvement in cellulose microfibril orientation. Plant Cell (2005) 17:1749–1763.
Schindelman G, Morikami A, Jung J, Baskin TI, Carpita NC, Derbyshire P, McCann MC, Benfey PN. COBRA encodes a putative GPI-anchored protein, which is polarly localized and necessary for oriented cell expansion in Arabidopsis. Genes Dev. (2001) 15:1115–1127.
Spring DR. Chemical genetics to chemical genomics: small molecules offer big insights. Chem. Soc. Rev. (2005) 34:472–482.[CrossRef][Medline]
Sugimoto K, Himmelspach R, Williamson RE, Wasteneys GO. Mutation or drug-dependent mictotubule disruption causes radial swelling without altering parallel cellulose microfibril deposition in Arabidopsis root cells. Plant Cell (2003) 15:1414–1429.
Sugimoto K, Williamson RE, Wasteneys GO. Wall architecture in the cellulose-deficient rsw1 mutant of Arabidopsis thaliana: microfibrils but not microtubules lose their transverse alignment before microfibrils become unrecognizable in the mitotic and elongation zones of roots. Protoplasma (2001) 215:172–183.[CrossRef][ISI][Medline]
Ueda K, Matsuyama T, Hashimoto T. Visualization of microtubules in living cells of transgenic. Arabidopsis thaliana. Protoplasma (1999) 206:201–206.
Upadhyaya MK, Noodén LD. Relationship between the induction of swelling and the inhibition of elongation caused by oryzalin and colchicine in corn roots. Plant Cell Physiol. (1978) 19:133–138.
Wasteneys GO. Progress in understanding the role of microtubules in plant cells. Curr. Opin. Plant Biol. (2004) 7:651–660.[CrossRef][ISI][Medline]
Wasteneys GO, Fujita M. Establishing and maintaining axial growth: wall mechanical properties and the cytoskeleton. J. Plant Res. (2006) 119:5–10.[CrossRef][ISI][Medline]
Whittington AT, Vugrek O, Wei KJ, Hasenbein NG, Sugimoto K, Rashbrooke MC, Wasteneys GO. MOR1 is essential for organizing cortical microtubules in plants. Nature (2001) 411:610–613.[CrossRef][Medline]
Williamson RE, Burn JE, Baskin TI, Arioli T, Betzner AS, Cork A. Morphology of rsw1, a cellulose-deficient mutant of Arabidopsis thaliana. Protoplasma (2001) 215:116–127.[CrossRef][ISI][Medline]
(Received August 14, 2007; Accepted September 12, 2007)
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