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American Society of Plant Biologists
The Arabidopsis TUBULIN-FOLDING COFACTOR A Gene Is Involved in the Control of the
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Abstract |
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- and 
-tubulin is important during microtubule biogenesis. This process involves several tubulin-folding cofactors (TFCs), of which only TFC A is not essential in mammalian in vitro systems or in vivo in yeast. Here, we show that the TFC A gene is important in vivo in plants. The Arabidopsis gene KIESEL (KIS) shows sequence similarity to the TFC A gene. Expression of the mouse TFC A gene under the control of the 35S promoter rescues the kis mutation, indicating that KIS is the Arabidopsis ortholog of TFC A. kis plants exhibit a range of defects similar to the phenotypes associated with impaired microtubule function: plants are reduced in size and show meiotic defects, cell division is impaired, and trichomes are bulged and less branched. Microtubule density was indistinguishable from that of the wild type, but microtubule organization was affected in trichomes and hypocotyl cells of dark-grown kis plants. The kis phenotype was rescued by overexpression of an -tubulin, indicating that KIS is involved in the control of the correct balance of - and -tubulin monomers. |
INTRODUCTION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSReferences |
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/-tubulin heterodimers (Mitchison and Kirschner, 1984
; Hirokawa, 1994; Mandelkow and Mandelkow, 1995).
The formation of assembly-competent /-tubulin heterodimers involves a sequence of chaperone-mediated steps (Lewis et al., 1997). After translation, both - and -tubulin monomers are captured by chaperonins of the GroEL and Hsp60 class named chaperonin c-cpn, which mediate correct protein folding (Rommelaere et al., 1993; Kubota et al., 1994). In vitro studies in mammalian systems suggest that -tubulin binds initially to tubulin-folding cofactor (TFC) B and -tubulin binds initially to TFC A. These are replaced subsequently by TFCs E and D, respectively. The two pathways converge to form a quaternary complex consisting of -tubulin, TFC E, -tubulin, and TFC D. The assembly-competent /-tubulin heterodimers are released from the complex upon binding of TFC C (Lewis et al., 1997). In addition, Arl2, a small G-protein, seems to play a regulatory role in sequestering TFC D (Bhamidipati et al., 2000).
This complex system not only produces free units for microtubule assembly but also is involved in the control of the correct concentration of /-tubulin heterodimers for microtubule formation and the balance between - and -tubulin monomers. The latter is particularly important, because an excess of free -tubulin was shown to be toxic and leads to lethality in yeast (Burke et al., 1989; Weinstein and Solomon, 1990).
Two lines of evidence suggest that TFC A functions mainly to maintain a reservoir of bound and nontoxic -tubulin monomers and thereby serves as a buffer protecting the cell from an unbalanced /-tubulin ratio. First, overexpressed -tubulin in yeast can be counteracted by overexpression of TFC A (Archer et al., 1995). Second, although TFC A binds to partially folded -tubulin in in vitro assays, it does not participate in the actual protein-folding reactions (Tian et al., 1996). The function of the TFC A gene is dispensable in budding yeast and in fission yeast under normal growth conditions (Archer et al., 1995; Radcliffe et al., 2000).
In plants, microtubules have an important function in chromosome separation, cell division, and the establishment of cell polarity (Baskin and Cande, 1990; Mayer et al., 1999; Mathur and Chua, 2000). In recent years, one major effort to study their in vivo role was a genetic approach in Arabidopsis. Several mutants have been identified in which the microtubule cytoskeleton generally is affected: fass/ton1, botero1, fragile fiber2, microtubule organization1, angustifolia, and spike1 (spk1) (Traas et al., 1995; Bichet et al., 2001; Burk et al., 2001; Whittington et al., 2001; Folkers et al., 2002; Kim et al., 2002; Qiu et al., 2002). In addition, the cloning of genes involved in the morphogenesis of one or several cell types revealed that they encode proteins with sequence similarity to known microtubule-associated factors.
The trichome morphogenesis gene ZWICHEL (ZWI) encodes a protein with sequence similarity to a kinesin motor molecule, and it is speculated that ZWI is involved in the local reorientation of microtubules during branch formation (Hülskamp et al., 1994; Reddy et al., 1996; Oppenheimer et al., 1997; Reddy and Day, 2000). The FRAGILE FIBER2 gene is important for cell elongation in various cell types and for trichome branching and encodes a protein with sequence similarity to katanin, a protein that regulates microtubule disassembly by severing microtubules (Burk et al., 2001). Mutations in the SPK1 gene lead to defects in polarized growth in all cotyledon and leaf epidermal cell types (Qiu et al., 2002). The SPK1 gene encodes a protein with a CED-5, DOCK180, MBC, SPK1 domain that is hypothesized to mediate cytoskeletal reorganization in response to diverse extracellular signals.
Mutations in genes that affect the biogenesis of microtubules result in lethal embryos that consist of one or few enlarged cells and lack microtubules (Mayer et al., 1999). After submission of this article, the PILZ group genes PORCINO, CHAMPIGNON, PFIFFERLING, and HALLIMASCH were reported to encode TFCs C, D, and E and Arl2, respectively, and the KIESEL (KIS) gene, with a related but weaker embryo-lethal phenotype, was shown to encode TFC A (Steinborn et al., 2002). Independently, Meinke and colleagues also isolated the genes encoding Arl2 (TITAN5 [McElver et al., 2000]) and TFC D (TITAN1 [Tzafrir et al., 2002]). These genes have been identified by mutations that result in severe cell division defects during endosperm and embryo development (Liu and Meinke, 1998; McElver et al., 2000).
In this work, we describe the cloning of the KIS gene, which encodes the Arabidopsis TFC A gene. We show that, in contrast to the mammalian in vitro system and the genetic data in yeast showing that TFC A is not essential, a weak mutation in the Arabidopsis TFC A gene is sufficient to cause severe cell morphogenesis and cell division defects. Our finding that all phenotypic aspects can be rescued by overexpression of an -tubulin indicates that TFC A has an important role in maintaining the balance between the /-tubulin monomers.
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RESULTS |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSReferences |
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; Szymanski et al., 1999). The function of KIS is not dependent on actin. To determine whether cell expansion is affected in kis-T1 mutants, we assessed root cortical cells and epidermal hypocotyl cells. Both cell types were indistinguishable from the wild type under normal growth conditions. When plants were challenged by growing them in the dark, which triggers rapid elongation of hypocotyl cells, we observed a marked difference compared with the wild type. Under normal light conditions, wild-type and kis-T1 mutant hypocotyls were similar in length (Figure 2A). However, in the dark, hypocotyl elongation was much reduced in kis-T1 mutants compared with the wild type (Figure 2D). Epidermal hypocotyl cells were bulged and short (Figure 2E), indicating that they had lost growth polarity.
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) and embryo sac development has proceeded to the binucleate stage, kis-T1 mutants still lacked an embryo sac (Figures 4E to 4H). These data indicate that in kis-T1 mutants, female meiosis is absent and that, on the male side, development is aborted at or immediately after meiosis.
KIS Encodes a TFC A Homolog
The kis-T1 mutant was isolated from a T-DNA population generated at the Institut National de la Recherche Agronomique (Versailles, France). Cosegregation analysis showed that the kis-T1 mutant phenotype cosegregated with the resistance marker of the inserted T-DNA in 128 homozygous mutant F2 plants. Therefore, we cloned the genomic DNA flanking the left border of the T-DNA insertion.
This genomic fragment maps to BAC T6B20 on chromosome 2 in the 5' region 32 bp upstream of the first exon of a gene annotated as the putative TFC A (Figure 5A). To verify that the KIS gene corresponds to the TFC A gene, we used a 3960-bp genomic fragment including 1738 bp of the 5' flanking sequence and a 1298-bp 3' flanking sequence of the TFC A–like gene for transformation into the kis-T1 mutant. All 12 transgenic plants recovered rescued all aspects of the kis-T1 mutant phenotype (data not shown).
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Alignment of the cDNA sequence with the genomic sequence indicated that the KIS gene has three exons. The deduced KIS gene product is 113 amino acids in length and shows 39% sequence identity and 52% sequence similarity to the mammalian TFC A gene and 
28% sequence identity and 45% sequence similarity to the Saccharomyces cerevisiae TFC A gene RBL2 (Figure 6). Arabidopsis database searches did not reveal any other gene with significant sequence similarity to the TFC A gene.
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Expression of Mouse TFC A Complements the kis Mutant
The sequence similarity of the KIS gene to the mammalian and yeast TFC A gene suggested that the KIS gene is involved in the correct folding of the -tubulin subunits. To test this possibility, we expressed the mouse TFC A (MmTFCA) gene and, as a control, the Arabidopsis KIS cDNA under the ubiquitous 35S promoter of Cauliflower mosaic virus in the kis mutant background. Both constructs rescued all aspects of the kis mutant phenotype. Rescued plants were fully fertile and indistinguishable in size from wild-type plants. For a comparison of organ size, we compared the leaf length and leaf width of the third leaf in 21 plants.
Compared with wild-type leaves (length, 7.3 ± 0.8 mm; width, 5.9 ± 0.6 mm), kis-T1 mutant leaves were reduced in size (length, 3.2 ± 0.3 mm; width, 2.2 ± 0.3 mm). Mutant kis-T1 plants transformed with 35S:KIS (length, 7.4 ± 0.8 mm; width, 6 ± 0.7 mm) and 35S:MmTFCA (length, 7.4 ± 0.9 mm; width, 6.1 ± 0.9 mm) were rescued to wild-type levels (Figure 3G). Subtle differences were found only with respect to the rescue of the branch phenotype of trichomes (Table 1). We did not observe any new phenotypic changes associated with overexpression of the KIS gene.
Overexpression of -Tubulin Can Rescue the kis-T1 Mutant Phenotype
The findings that in yeast the overexpression of TFC A (Rbl2p) can counteract the overexpression of -tubulin and, conversely, that the overexpression of TFC A can compensate for a quantitative defect in -tubulin indicate that TFC A is involved in the control of the /-tubulin monomer balance (Archer et al., 1995). To determine whether TFC A has a similar function, we tested the possibilities that overexpression of -tubulin might suppress and overexpression of -tubulin might enhance the kis phenotype. The Arabidopsis cDNAs of - and -tubulin were cloned under the control of the 35S promoter, and the constructs were inserted into the kis mutant background. We found no phenotypic enhancement of the kis phenotype with -tubulin. However, rescue of all phenotypic aspects was found in plants overexpressing -tubulin. Only trichome branching was not rescued completely (Table 1).
Microtubule Organization Is Disturbed in kis-T1 Mutants
Analogous with mammalian systems and yeast, the reduction or the absence of TFC A should lead to reduced levels of properly folded -tubulin and, as a consequence, to reduced levels of assembly-competent /-tubulin heterodimers. One might expect that this would also result in fewer microtubules and/or in different microtubule behavior, because changes in the concentration of free dimers should change microtubule dynamics.
To study microtubule density and organization in vivo, we introduced the 35S::MAP4-green fluorescent protein (GFP) construct in the kis-T1 mutant background. The MAP4-GFP protein binds to microtubules and thereby labels them (Marc et al., 1998). We focused on the analysis of the microtubule organization in hypocotyl cells under different growth conditions and in trichomes because their size facilitates the observation and because they undergo characteristic changes during development, thus providing criteria to assess their behavior. Microtubule density in the kis mutant was similar to that in the wild type (Figures 7A and 7B).
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), this phenotype could reflect a failure to polymerize new microtubules as a result of a reduced availability of assembly-competent /-tubulin heterodimers.
During trichome development, cortical microtubules are arranged initially transversally and shift to a longitudinal orientation after branch formation (Mathur and Chua, 2000). In contrast to the situation in the wild type, we frequently found trichomes in kis-T1 mutants displaying transversally oriented cortical microtubules (Figures 7A and 7B).
Microtubules are important for the initiation of branching, and it has been shown that in the underbranched trichome mutant sti, branch initiation can be stimulated by the transient stabilization of microtubules (Mathur and Chua, 2000). Thus, the ability to induce branch formation by microtubule stabilization should provide some insights into microtubule properties. We compared the effect of transient taxol treatments on branch initiation in sti and kis-T1 mutants. The microtubule-stabilizing drug taxol was applied to young plants for a limited time and washed out to allow recovery.
In sti mutants, which normally are unbranched (100% unbranched; n = 400), this treatment induced one additional branch in 19% and two additional branches in 1% of the trichomes (n = 314) (Figure 1M). In kis-T1 mutants, no additional branch formation was observed after this treatment (Figures 1N and 1O). In untreated plants, 93% were unbranched and 7% of trichomes had one branch point (n = 272); in taxol-treated plants, 96% were unbranched and 4% had one branch point (n = 273). It should be noted that the difference in branch number in these experiments compared with soil-grown plants is the result of the in vitro growth conditions. Thus, in contrast to the situation in sti mutants, branch induction by taxol is ineffective in kis-T1 mutants.
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DISCUSSION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSReferences |
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; Mandelkow and Mandelkow, 1995). On the other hand, microtubule function depends critically on factors that coordinate their biogenesis and recycling of disassembled monomers and dimers of tubulin (Lewis et al., 1997). TFC A has an important role in regulating the balance of - and -tubulin monomers (Archer et al., 1995; Tian et al., 1996; Radcliffe et al., 2000). The findings that, in contrast to yeast, strong mutations in Arabidopsis TFC A result in embryo lethality (Steinborn et al., 2002) and weak mutations result in severe postembryonic defects in cell division and cell morphogenesis (this study) provide a glimpse of the regulatory differences of the in vivo role of TFCs between yeast and higher eukaryotes.
KIS Is the Arabidopsis Ortholog of the Mouse TFC A Gene
The kis-T1 allele we have identified contains a T-DNA insert in the promoter region that stimulates the transcription of a new transcript containing the open reading frames of the BAR gene located on the T-DNA and the TFC A gene. Therefore, it is likely that the protein level is reduced, because translation of the TFC A gene can occur only as a result of reinitiation of translation. The fact that the kis-T1 phenotype is rescued by a genomic fragment proves that the KIS gene encodes the Arabidopsis TFC A gene and excludes the possibility that the kis-T1 phenotype is caused by some kind of dominant effect that results from misexpression attributable to the T-DNA insertion in the promoter region. A sequence identity of 39% between the biochemically well-characterized mammalian TFC A gene (Tian et al., 1996) and the Arabidopsis TFC A gene suggests that they have the same biochemical functions. Our finding that the mouse TFC A gene can rescue the Arabidopsis TFC A mutant clearly shows that the mouse TFC A gene can functionally replace the Arabidopsis TFC A gene.
Function of TFC A in Arabidopsis
The biochemical function of TFC A was studied in mammalian in vitro systems (Tian et al., 1996). It was shown that TFC A binds to -tubulin intermediates derived from the chaperonin c-cpn and that, in a next step, -tubulin is transferred to TFC D (Melki et al., 1996; Tian et al., 1996). However, TFC A is not required for this step in vitro, because exchange-competent -tubulin also was formed in the absence of TFC A. Therefore, it was suggested that TFC A functions as a reservoir of c-cpn–generated intermediates pending their transfer to TFC D.
Consistent with its accessory function, TFC A is the only cofactor not essential in budding and fission yeast (Archer et al., 1995; Radcliffe et al., 2000). Thus, it was surprising that TFC A is essential in Arabidopsis to the extent that strong alleles lack microtubules and lead to embryo lethality (Steinborn et al., 2002), and the weak allele analyzed in this study shows a range of phenotypes that are similar to the defects caused by microtubule malfunctioning (Baskin et al., 1994; Mayer et al., 1999; Mathur and Chua, 2000). One possible explanation for this effect is that TFC A has adopted completely new functions in plants.
This is unlikely, however, because the biochemical properties are not much altered, as is evident from the rescue of the TFC A phenotype by the mouse TFC A gene. In addition, our finding that mutations in the Arabidopsis TFC A gene can be rescued by the overexpression of -tubulin further supports the idea that Arabidopsis TFC A has a similar role in the tubulin-folding pathway, as found in the mammalian and yeast systems. Therefore, it is conceivable that in Arabidopsis, the requirements to capture the partially folded -tubulin released from the chaperonin and transfer it to TFC D have become more demanding.
Insights into Microtubule Behavior
Because the TFC A gene acts at an early step in microtubule biogenesis, it is likely that the observed defects in cell morphogenesis and cell division in the kis-T1 mutant are caused not by changes in biochemical properties of microtubules but by a reduction in microtubule levels. However, microtubule density was not affected notably in the kis-T1 mutant, suggesting that defects in microtubule function are caused by a reduced availability of free /-tubulin heterodimers and most likely altered microtubule dynamics.
Because it has been reported previously that microtubule reorientation in plants during cell division and cell growth involves the disassembly of existing microtubules and their de novo assembly (Hush et al., 1994; Yuan et al., 1995), it is conceivable that the observed phenotypes in kis-T1 mutants can be explained in this way. The failure to reorient cortical microtubules in the kis-T1 mutant was seen very clearly in trichomes, in which cortical microtubules remained in their initial transverse orientation and did not shift to a longitudinal orientation after branch formation. Similarly, the inability of kis-T1 mutants to maintain a transverse orientation of microtubules in dark-grown hypocotyl cells suggests that the reorganization of microtubules under rapid growth conditions cannot be achieved in a timely manner.
Also, the observed defects in establishing a new growth axis (as in branch initiation) in trichomes can be interpreted in this manner. The previous finding that the transient stabilization of microtubules by taxol can induce branch formation suggests that the correct timing of microtubule assembly and disassembly is important for branch formation (Mathur and Chua, 2000). Our finding that this treatment does not induce new branches in kis-T1 mutants suggests that the manipulation of microtubule dynamics by taxol is rendered inefficient because the reservoir of free /-tubulin heterodimers is reduced.
Perspective
Although the functional characterization of the Arabidopsis TFC genes (Steinborn et al., 2002; Tzafrir et al., 2002) indicates that the biogenesis of microtubules involves the same set of factors as in other organisms, some important differences become apparent that suggest that their relative contribution and importance in microtubule function are different. A more detailed genetic, molecular, and biochemical analysis of microtubule biogenesis will be important to understand the control of microtubule organization and function.
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METHODS |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSReferences |
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Transient taxol treatments were performed using 2-week-old plants. Plants were treated for 30 min with 20 µM taxol dissolved in Murashige and Skoog (1962) medium and washed five times with Murashige and Skoog (1962) medium. Phenotypes were assessed after 3 days. Agrobacterium tumefaciens–mediated transformation of Arabidopsis plants was performed as described by Clough and Bent (1998). Microtubule organization was studied using a transgenic MAP4:green fluorescent protein line (Mathur and Chua, 2000).
Nucleic Acid Analyses
To identify the T-DNA–flanking genomic sequences, Vectorette PCR was applied (Koncz et al., 1992). No amplification products were obtained for the right T-DNA border. The genomic region flanking the left T-DNA border was identified using the restriction enzymes BfaI and MboI and the following primers: TLB-0 (5'-AGACAACCCTCAACTGGAAACG-3'), TLB-1 (5'-TGTGCCAGGTGCCCACGGAAT-AG-3'), VEC-1 (5'-CGAATCGTAACCGTTCGTACGAGAA-3'), and VEC-2 (5'-TCGTACGAGAATCGCTGTCCTCTCC-3'). The PCR fragment was subcloned into pGEM-T (Promega, Madison, WI) and sequenced. For genomic rescue of the kis-T1 mutants, a 3960-bp genomic HpaI-SpeI fragment of BAC T6B20 was cloned into pBluescript KS+ (Stratagene, La Jolla, CA) and subcloned into pGPTV-HPT (Becker et al., 1992).
Total RNA was extracted as described by Heim et al. (1993). Fifteen micrograms of total RNA from plants at the rosette stage was used for RNA gel blot analysis. Prehybridization, hybridization, and detection were performed as described by Sambrook et al. (1989). Equal loading was controlled by rehybridization of the filter with the 26S rRNA probe. To analyze KIS gene expression by reverse transcriptase–mediated (RT) PCR, total RNA was isolated from different organs and treated with DNase using the DNA-free kit (Ambion, Austin, TX) before first-strand cDNA synthesis.
First-strand cDNA was synthesized from 1 µg of RNA using Superscript II Reverse Transcriptase (Invitrogen, Groningen, The Netherlands) according to the manufacturer's instructions. The KIS cDNA was amplified using the following primers: EAL-s1 (5'-GGATCCTAC-AATGGCAACGATAAGGAAC-3') and EAL-as1 (5'-GGTACCGATTTA-ACACTCATCGCTG-3'). For semiquantitative RT-PCR, minimal numbers of cycles, which resulted in a visible band on an agarose gel, were used to ensure that amplifications were within the linear range. One microliter of first-strand synthesis reaction was amplified under the following conditions: 94°C for 2 min followed by 25 cycles of 94°C for 20 s, 56°C for 30 s, 72°C for 30 s, and 72°C for 5 min. As a control, the ubiquitously expressed Arabidopsis EF1A4 gene was amplified for 25 cycles using the primers EF1A4-UP and EF1A4-RP designed by Nesi et al. (2000).
For detection of the chimeric BAR-AtTFCA transcript, the BAR-s2 primer (5'-CTGCACCATCGTCACCACTAC-3') was used in combination with the EAL-as1 primer (94°C for 2 min and then 30 cycles of 94°C for 20 s, 58°C for 30 s, 70°C for 3 min, and 72°C for 7 min). To exclude contamination of the cDNA with genomic DNA, control PCR with primers located at the 3' end of the 35S promoter (35S-k14, 5'-GACGTTCCAACCACGTCTTC-3') and in the KIS gene (EAL-as1) was included.
Vectors and Constructs
To construct the 35S::KIS transcriptional fusion, a full-length cDNA of KIS was amplified by RT-PCR using proofreading Pfu polymerase (Stratagene, La Jolla, CA) and the primers EAL-s1 and EAL-as1 and cloned into the BamHI and KpnI endonuclease recognition sites of the pGEM-T vector (Promega). The cDNA fragment was subcloned into the binary vector p35S-HPT, which was created by inserting an EcoRI-HindIII fragment containing the 35S promoter and transcription terminator of pBinAR (Höffgens and Willmitzer, 1990) into pGPTV-HPT (Becker et al., 1992).
To create the 35S::MmTFCA, a mouse tubulin-folding cofactor (TFC) A cDNA fragment (Llosa et al., 1996) was excised as a XbaI-HindIII fragment from the pET3a vector (Novagen, Madison, WI) and inserted into p35S-HPT vector digested with XbaI and SalI. HindIII and SalI sites were filled with Klenow DNA polymerase. To create rescue constructs with Arabidopsis - and -tubulin, 4-tubulin and 5-tubulin cDNA fragments were excised from the pBS-TUA4 and pBS-TUB5 vectors (kindly provided by Bernd Geiges, University of Tübingen) as KpnI-XbaI and SmaI-XhoI fragments, respectively, and inserted into p35S-HPT.
Microscopy
Scanning electron microscopy was performed as described previously (Adler et al., 1996). 4',6-Diamidino-2-phenylindole and aniline blue staining were performed as described previously (Hülskamp et al., 1997). For whole-mount ovule preparations, siliques were dissected and fixed on ice in ethanol:distilled water:acetic acid:37% formaldehyde (10:7:2:1) for 30 min, hydrated in a graded ethanol series to 50 mM NaPO4 buffer, pH 7.2, and mounted on microscope slides in a clearing solution of chloral hydrate:water:glycerol (8:2:1).
Confocal laser scanning microscopy was performed using the Leica TCS SP2 system (Wetzlar, Germany). Images were processed using Adobe Photoshop 6.0 (Mountain View, CA) and Aldus Freehand 7.0 (Seattle, WA) software.
Upon request, all novel materials described in this article will be made available in a timely manner for noncommercial research purposes. No restrictions or conditions will be placed on the use of any materials described in this article that would limit their use for noncommercial research purposes.
Accession Numbers
The GenBank accession numbers for the genes mentioned in this article are X16432 (Arabidopsis EF1A4), M84697 (4-tubulin), and M84702 (5-tubulin). The AGI number for the putative TFC A is At2 g30410.
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Acknowledgments |
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4-tubulin and 5-tubulin cDNA. We thank Nam-Hai Chua for making available the MAP4:green fluorescent protein line. We thank members of our laboratory for critically reading the manuscript. This work was supported by a grant to M.H. from the Volkswagen Stiftung. |
Footnotes |
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Received March 12, 2002; accepted May 17, 2002.
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSReferences |
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-Tubulin Monomer Balance
