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First published online November 19, 2004; 10.1104/pp.104.051748 Plant Physiology 136:3933-3944 (2004) © 2004 American Society of Plant Biologists
Plant-Specific Microtubule-Associated Protein SPIRAL2 Is Required for Anisotropic Growth in Arabidopsis1Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma 630–0192, Japan (T.S., N.N.N., J.A., K.N., T. Hashimoto); and Department of Life Science, Faculty of Science, University of Hyogo, Harima Science Park City, Hyogo 678–1297, Japan (T. Hamada, S.S.)
In diffusely growing plant cells, cortical microtubules play an important role in regulating the direction of cell expansion. Arabidopsis (Arabidopsis thaliana) spiral2 (spr2) mutant is defective in directional cell elongation and exhibits right-handed helical growth in longitudinally expanding organs such as root, hypocotyl, stem, petiole, and petal. The growth of spr2 roots is more sensitive to microtubule-interacting drugs than is wild-type root growth. The SPR2 gene encodes a plant-specific 94-kD protein containing HEAT-repeat motifs that are implicated in protein-protein interaction. When expressed constitutively, SPR2-green fluorescent protein fusion protein complemented the spr2 mutant phenotype and was localized to cortical microtubules as well as other mitotic microtubule arrays in transgenic plants. Recombinant SPR2 protein directly bound to taxol-stabilized microtubules in vitro. Furthermore, SPR2-specific antibody and mass spectrometry identified a tobacco (Nicotiana tabacum) SPR2 homolog in highly purified microtubule-associated protein fractions from tobacco BY-2 cell cultures. These results suggest that SPR2 is a novel microtubule-associated protein and is required for proper microtubule function involved in anisotropic growth.
The microtubule is a dynamic filamentous polymer consisting of heterodimers of highly conserved  - and  -tubulin subunits and plays key roles in cell morphogenesis, polarity establishment, cell division, intracellular organelle transport, and other cellular processes in all eukaryotic cells (Desai and Mitchison, 1997 ). Despite the conserved polymer structure of microtubules, the organization and dynamics of microtubule arrays are remarkably diverse and change during the cell cycle, in different cell types, and in response to environmental and developmental stimuli. As the plant cell cycle progresses, microtubules are organized into the preprophase band, the mitotic spindle, the cytokinetic phragmoplast, the perinuclear array, and the cortical array (Wasteneys, 2002). Cortical microtubule arrays in interphase cells are arranged in a random meshwork or adopt a more ordered distribution (Cyr and Palevitz, 1995). In rapidly elongating cells with diffuse growth, cortical arrays are arranged perpendicular to the cell's growth axis and are thought to dictate the growth anisotropy (Sugimoto et al., 2000). Physical stimuli, such as gravity and light, and plant hormones are known to rearrange cortical microtubule arrays within an hour from one predominant orientation to another (Shibaoka, 1994; Wymer and Lloyd, 1996).
The dynamics and array organization of microtubules are likely controlled by stabilizing and destabilizing factors, as well as other microtubule regulators (Hashimoto, 2003
Molecular genetic analyses of helical growth mutants of Arabidopsis have been contributing to our understanding of anisotropic growth regulation. Epidermal cells of longitudinally elongating tissues in these twisted mutants are skewed and, importantly, in several helical growth mutants the orientation of helices is fixed in either a right- or a left-handed direction (Hashimoto, 2002
spiral2 (spr2) has been isolated as an Arabidopsis right-handed helical mutant with prominent counterclockwise twisting in leaf petioles and flower petals, when viewed from above (Furutani et al., 2000 In this study, we cloned the SPR2 gene using a map-based strategy. SPR2 is a member of a thus far uncharacterized HEAT-repeat-containing protein family and binds to microtubules both in vivo and in vitro. We demonstrate that a defect in SPR2 MAP results in a right-handed helical growth phenotype.
Phenotypes and Sensitivity to Microtubule-Interacting Drugs
We have previously isolated spr2-1 in the Landsberg erecta (Ler) background and reported that spr2 is allelic to a classic twisting mutant convoluta (Furutani et al., 2000
To test whether spr2 is compromised in terms of microtubule function, we examined the effects of microtubule-interacting drugs on the growth of primary roots. When grown on a vertically placed hard agar plate, wild-type roots (Ler ecotype) grew slightly toward the left side of the plate, whereas spr2 roots grew slightly toward the right (Fig. 1E), indicating that the spr2 mutation affected the root as well. When seedlings were grown on a medium containing a low dose of the microtubule-depolymerizing drug oryzalin (0.1 µM) or the microtubule-stabilizing drug taxol (1 µM), growth of wild-type primary roots was inhibited slightly by oryzalin and by 25% by taxol (Fig. 1F). At these drug concentrations, spr2 roots were affected somewhat more strongly than wild-type roots.
To substantiate the drug response, we studied the effect of the spr2 mutation in the lefty2 tubulin mutant background (Thitamadee et al., 2002
By crossing spr2-1 with wild-type Col, the SPR2 locus had been mapped between molecular markers PG11 and mi123 on chromosome 4 (Furutani et al., 2000
The 1-bp deletion in the third exon of At4g27060 in spr2-1 would cause a frameshift in the encoded protein after amino acid 550. Further sequence analysis revealed that the other three spr2 alleles all carried mutations in this gene. In spr2-2, the 5' exon-intron boundary of the third exon was changed from AG:AT to AA:AT. This change destroys the splicing consensus at the acceptor site. In spr2-3, the C at 1,585 was changed to a T, resulting in the substitution of Glu-528 with a stop codon. We also found a 2-bp deletion in the first exon of At4g27060 in the convoluta allele, which would cause a frameshift after amino acid 76. A line with a T-DNA insertion in the third exon of At4g27060 (designated as spr2-4) was identified in the SALK T-DNA collection and showed a right-handed helical growth phenotype characteristic of spr2 mutants when the T-DNA was present in the homozygous state. These five spr2 alleles showed very similar phenotypes, indicating that these are all null alleles. A full-length cDNA of At4g27060 was assembled, fused to 1.3-kb 5'-flanking and 1.4-kb 3'-flanking genomic regions, and introduced into spr2-2 by an Agrobacterium-mediated transformation method. Thirteen kanamycin-resistant transgenic lines were generated and clear complementation of the spr2 mutant phenotype was confirmed in 11 of them (Fig. 2C). We therefore conclude that At4g27060 is the SPR2 gene.
SPR2 is predicted to encode a protein of 864 amino acids with an estimated molecular mass of 94 kD and a pI of 5.47 (Fig. 2B). A BLAST search of databases identified similar proteins of unknown function in Arabidopsis, potato (Solanum tuberosum), and rice (Oryza sativa; Fig. 3A
) and multiple expressed sequence tag sequences in various plants, but no homologous proteins in nonplant organisms. The Arabidopsis protein encoded by At1g50890 and potato HIP2 share the highest similarity to SPR2 over the entire length (48% and 50% identity to SPR2, respectively; Fig. 3A). Potato HIP2 had been isolated as a host protein interacting with a potyviral multifunctional protein helper-component proteinase by a yeast (Saccharomyces cerevisiae) two-hybrid screening (Guo et al., 2003
The extreme N-terminal region (amino acids 1–37) of SPR2 is rich in Ser and Thr (11 and 6 out of 37 residues, respectively; Fig. 3B). A Ser/Thr-rich N terminus was also found in At1g50890 and HIP2 but not in other homologs. A motif search with the PSORT program (http://psort.nibb.ac.jp) predicted that this N-terminal region is a targeting signal to plastids, but cellular localization studies (see below) demonstrated that SPR2 is not targeted to plastids.
We did not detect significant levels of the SPR2 transcript in wild-type plants by RNA gel-blot analysis. The low abundance of SPR2 sequences in the Arabidopsis expressed sequence tags and massively parallel signature sequencing databases (http://www.arabidopsis.org/Blast/ and http://mpss.udel.edu/at) supports that the SPR2 expression level is low in wild-type Arabidopsis plants. Therefore, we carried out a reverse transcription (RT)-PCR analysis using primers specific to SPR2 but not to its homologs in Arabidopsis. SPR2 expression was detected in flower bud, cauline leaf, rosette leaf, inflorescence stem, root, and cotyledon at similar levels (Fig. 4A ), indicating that SPR2 expression is not restricted to any particular organ.
Next, we raised an anti-SPR2 polyclonal antibody against an N-terminal region of recombinant SPR2 and used it to detect SPR2 protein in Arabidopsis seedlings (Fig. 4B). The antibody recognized a band of about 90 to 95 kD in wild-type seedlings but not in spr2-2 seedlings, indicating its specificity. In wild-type seedlings, more SPR2 protein was recovered in the microsomal fraction than in the soluble fraction.
Since the expression level of SPR2 appears to be low in wild-type plants, we examined the consequences of overexpression for cell morphology and growth. SPR2 was fused to a tandem tag of hemaglutinin and hexa-His at its C terminus and expressed under the transcriptional control of the cauliflower mosaic virus (CaMV) 35S promoter in Arabidopsis plants. When the fusion protein was expressed in spr2-2, the helical mutant phenotype was effectively complemented with the transgene expression (data not shown), indicating that this SPR2 fusion protein is functional in plants. We transformed wild-type Wassilewskija plants with the construct and obtained 18 kanamycin-resistant transgenic lines. Ten lines showed right-handed helical growth characteristic of the spr2 mutant, while the remaining eight lines did not differ from wild-type plants in growth or cell morphology and did not show any twisting phenotypes (Fig. 5A ). Two lines each from the twisting and wild-type growth groups were analyzed further. In both twisting lines, expression levels of SPR2 were severely reduced, probably by cosuppression (Fig. 5B). In contrast, SPR2 expression was considerably increased in the two lines that showed wild-type growth (Fig. 5B). The SPR2 protein level was also highly increased in these lines (see Fig. 4B). These results indicate that SPR2 overexpression does not induce helical growth or other morphological abnormalities.
SPR2-GFP Protein Associates with Microtubules in Vivo To examine the intracellular localization, SPR2 was fused to GFP at its C terminus and was expressed under the transcriptional control of the CaMV 35S promoter. When expressed in spr2-2, the SPR2-GFP fusion gene rescued the spr2 mutant phenotype (data not shown), suggesting the functional integrity of this fusion protein. Confocal microscopic analysis revealed that SPR2-GFP fluorescence aligned along filamentous structures in the cortical region of leaf epidermal cells (Fig. 6A ). When the epidermal cells were optically sectioned at deeper focal planes, no obvious fluorescent structures were observed in the cytoplasmic regions, but strong fluorescent dots were seen underneath the plasma membrane (Fig. 6B). At high magnification, the cortical filamentous labeling was found in either continuous or linear punctuate patterns (Fig. 6C). In a single cell, both patterns are often present in varying preference to either pattern. Similar filamentous labeling was also observed in leaf trichome (Fig. 6D), epidermal cells of etiolated hypocotyl (Fig. 6E), and epidermal cells of the differentiated region in root (Fig. 6F).
The cortical filamentous labeling suggests an association of SPR2-GFP with cortical microtubules. To confirm this, we treated the leaf tissues with the microtubule-destabilizing drug propyzamide (Fig. 6G). This treatment caused the disappearance of filamentous fluorescent patterns, leaving fluorescent foci at the cell cortex. Similar results were obtained with oryzalin (data not shown). In addition to cortical microtubules, SPR2-GFP was also localized to mitotic spindles and phragmoplast microtubules in dividing cells of the transgenic Arabidopsis roots (Fig. 6H). Cortical microtubules, preprophase bands, mitotic spindles, and phragmoplasts were also stained with SPR2-GFP in stably transformed tobacco BY-2 cells (data not shown).
To test whether SPR2 directly binds to microtubules, we carried out an in vitro microtubule-cosedimentation assay. Full-length SPR2 was fused at the N terminus with thioredoxin and poly-His tags (TH-SPR2) and was expressed in Escherichia coli. Chromatography and the cleavage of N-terminal tags with a protease resulted in a homogeneous preparation of recombinant SPR2 in a native form (Fig. 7A ). Taxol-stabilized microtubules were prepared from MAP-free tubulins of tobacco BY-2 cells. When the purified SPR2 was centrifuged in the absence of microtubules, the protein remained in the supernatant. However, a significant proportion of SPR2 was recovered in the pellet when it was incubated with an excess of microtubules (Fig. 7B).
To determine the stoichiometry and affinity of SPR2 toward microtubules, various amounts of SPR2 were centrifuged with a constant quantity of microtubules, and a binding curve was obtained (Fig. 7C). Recombinant SPR2 bound to microtubules in a concentration-dependent and saturable manner. The binding to microtubules was saturated at a stoichiometry of 0.26 ± 0.02 mol of SPR2 per mole of tubulin dimer if we assume that SPR2 binds uniformly to microtubule side walls. The dissociation constant Kd of SPR2 was determined to be 0.23 ± 0.04 µM.
To examine whether endogenous SPR2-like protein is associated with microtubules in plants other than Arabidopsis as well, we searched for the presence of a tobacco SPR2 homolog in purified MAP fractions. Vacuole-free miniprotoplasts were prepared from cultured tobacco BY-2 cells, and microtubules were purified from the protein-rich protoplast extracts through two rounds of microtubule polymerization and depolymerization using taxol (Igarashi et al., 2000 To unambiguously identify this SPR2-related protein, tobacco proteins in the purified MAP fraction were further fractionated by anion-exchange chromatography, and eluted fractions were analyzed by general protein staining and immunoblotting. Tobacco SPR2-related protein was eluted right after the MAP-65 and MAP-190 fractions (Fig. 8B) and before the tubulin fractions (data not shown). The amount of this protein appeared to be less than that of MAP-65 or MAP-190. The SPR2-related protein bands in fractions 48 to 50 were excised from the polyacrylamide gel and then subjected to liquid chromatography (LC)-tandem mass spectrometry (MS/MS) analysis. When the mass spectrum data were used to screen the National Center for Biotechnology Information (NCBI) nonredundant protein database, the best fit was found in the amino acid sequence of potato HIP2, a closely related homolog of SPR2. Six peptide sequences matched perfectly with the expected peptide sequences from tryptic digests of HIP2 (Figs. 8C and 3B). Taken together, we conclude that a tobacco SPR2 homolog was copurified with microtubules from tobacco BY-2 cells.
SPR2 Binds to Microtubules in Vivo and in Vitro In this study, we showed that SPR2-GFP fusion protein decorated microtubules in Arabidopsis cells and that recombinant SPR2 bound to taxol-stabilized microtubules in vitro. These results establish that SPR2 is a bona fide MAP from plants. In addition, tobacco SPR2 homolog was concentrated in MAP fractions highly purified from tobacco BY-2 cells.
When SPR2-GFP was overexpressed in Arabidopsis and tobacco cells, it decorated not only cortical microtubules but also preprophase bands, spindle microtubules, and phragmoplast microtubules. In such microtubule structures, we did not find any prominent labeling of either plus or minus ends of microtubules. Thus, SPR2 may not distinguish between the distinct microtubule structures found in interphase and mitotic cells and may not be recruited to microtubule ends. However, the data on the distribution of SPR2-GFP should be interpreted with caution. SPR2 transcripts are not abundant in Arabidopsis plants, and tobacco SPR2 homolog is present in relatively small amounts in purified MAP fractions from tobacco BY-2 cells, compared to major MAPs such as TMBP200, MAP-190, and MAP-65. Although the complementation of spr2 mutant phenotypes by overexpressed SPR2-GFP indicates that a fraction of the expressed fusion protein should function at normal cellular target sites, excess SPR2 molecules might associate with microtubule structures and microtubule regions for which endogenous SPR2 normally shows low affinity and may thus mask the true in vivo localization patterns. When mammalian plus-end labeling MAP CLIP170 was expressed transiently in cowpea protoplasts or stably in tobacco BY-2 cells as a construct fused to GFP, microtubules were labeled along their entire length as expression levels increased (Dhonukshe and Gadella, 2003
We observed that, in some cells, SPR2-GFP accumulated in discrete punctate patterns along the microtubules. Our time-lapse observation indicates that these SPR2-GFP dots on microtubules do not represent plus ends of microtubules (data not shown). Interestingly, a similar linear punctate labeling of microtubules was sometimes observed for the GFP fusion of an Arabidopsis end-binding protein, AtEB1a (Chan et al., 2003
SPR2 and its homologs contain tandemly repeated sequences that show characteristics of the HEAT motif. HEAT repeats are found in a wide variety of eukaryotic proteins, including those from which it derives its name (Huntingtin, elongation factor 3, a subunit of protein phosphatase 2A, and lipid kinase TOR1; Andrade and Bork, 1995
It is noteworthy that HEAT repeats also occur in two classes of proteins that associate with tubulins or microtubules. First, XMAP215/TOG (Ohkura et al., 2001 Because SPR2 does not contain any known microtubule-binding domains found in established MAPs, it may be recruited to microtubules mainly through the HEAT-repeat motifs. In vitro binding stoichiometry data indicate that one SPR2 molecule spans four tubulin dimers when bound to microtubules if it binds uniformly to microtubule side walls. Mutational analysis of the SPR2 sequence should reveal the significance of the HEAT repeat in microtubule binding in vitro and in vivo.
Alternatively, but not mutually exclusively, the SPR2 HEAT repeat may be used as a scaffold to interact with molecules other than microtubules. A potato SPR2 homolog HIP2 has been isolated as an interaction partner of potyvirus helper-component proteinase (HCpro; Guo et al., 2003
Mutations in the SPR2 gene cause right-handed helical growth in epidermal cells of elongating tissues, most notably in hypocotyls, petioles, and petals. Now that SPR2 is found to be a MAP, the mutant phenotype is understood in the context of microtubule functions. It should be noted, however, that we are yet to directly demonstrate microtubule abnormalities in spr2 mutants. This is partly because spr2 mutants show relatively mild defects in growth anisotropy, especially in the root where a strict relationship between the orientation of cortical microtubule arrays and direction of cell expansion is observed in the rapid elongation zone (e.g. Sugimoto et al., 2000
Nevertheless, moderate defects of microtubule function in spr2 can be inferred from the enhanced inhibition of root growth by microtubule-interacting drugs in the spr2 mutant background (Fig. 1F). The expression of a GFP-
lefty tubulin mutants (Thitamadee et al., 2002
Plant Growth and Genetic Crosses
Seedlings of Arabidopsis (Arabidopsis thaliana) were grown on agar plates as described (Furutani et al., 2000 For allelism tests, the newly isolated spr2-2 and spr2-3 were crossed with spr2-1, and phenotypic complementation was confirmed in the F1 seedlings. The double mutant between spr2-1 and lefty2 was selected in F2 populations according to their molecular lesions, and the F3 plants were used for analysis. For mapping, spr2-1 (Ler) was crossed with a wild-type plant (Col) and selfed to obtain an F2-mapping population.
Gross morphology was observed using an Olympus stereoscope SZX12 equipped with a digital camera DP70 or DP10 (Olympus, Tokyo). We used a replica method to observe epidermal cell files by scanning electron microscopy. Molds were made by pressing the tissues into polyvinylsiloxane impression material (Extrude, Kerr, Romulus, MI) and were filled with epoxy glue (Araldite, Ciba-Geigy, Research Triangle Park, NC). After being coated with platinum, the replica images were examined with an S4700 scanning electron microscope (Hitachi, Tokyo). Confocal imaging was performed with a Zeiss Axioplan microscope equipped with a confocal head LSM510 (Zeiss, Jena, Germany) and an argon ion laser. GFP was excited at a 488-nm laser line, and emitted fluorescence was collected through a 525/30 bandpass filter.
spr2-1 homozygous seedlings were selected from the F2-mapping population and their DNA was used to map the SPR2 locus using cleaved-amplified polymorphic sequence and simple sequence length polymorphic markers (http://www.arabidopsis.org/aboutcaps.html). Two PCR-based markers were newly designed to detect polymorphisms between Col and Ler near the SPR2 locus. Marker names, primer sequences, restriction enzymes used, and the ecotype cut by the enzyme are as follows: F10M23.1, 5'-CAAGTCTTCAACAGATGATG-3', 5'-CCACATCTAACCCACTAGAC-3', no restriction (simple sequence length polymorphic marker); and M4I22.1, 5'-CCGTTCTCTAATTGCATGTC-3', 5'-AGCATTTTGTTGACCGACTC-3', MboI, Ler. Candidate genes in the fine-mapped region were amplified from genomic DNA of Ler and spr2-1 by PCR and were directly sequenced to detect mutations. Both ends of SPR2 cDNA were determined using 5'- and 3'-RACE systems (Gibco BRL, Cleveland). Full-length SPR2 cDNA was cloned from Col wild-type seedlings by RT-PCR using the SuperScript First-Strand Synthesis System (Gibco BRL). From BAC clone T24A18, the 5'-flanking region (1,340 bp), and the 3'-flanking region (1,436 bp) of SPR2 were cloned by PCR. The cloned fragments were sequenced to verify that there were no unexpected mutations.
For pBI-SPR2, SPR2 cDNA was placed between the 5'- and 3'-flanking regions of SPR2 in pBI 101 (CLONTECH Laboratories, Palo Alto, CA). For pBI-35S-SPR2HH, SPR2 cDNA with a 3'-terminal tag (encoding N-GGLVGGYPYDVPDYAGVEHHHHHH-C) was subcloned immediately downstream of the CaMV 35S promoter in pBI121 (CLONTECH Laboratories). For pBI-SPR2-GFP, SPR2 cDNA was subcloned with a linker (N-GGLVRPPAGRGGGGAT-C) in-frame just upstream of GFP in pTH-2 (Niwa, 2003 Total RNA was isolated using the RNeasy kit (Qiagen, Valencia, CA) and converted to cDNA with the SuperScript First-Strand Synthesis System (Gibco BRL). For semiquantitative RT-PCR, we calibrated the amounts of cDNA templates by comparing the amounts of PCR products from a dilution series of the templates. Aliquots of first-strand cDNA were amplified by PCR for 3 min at 94°C, followed by 30 cycles of 30 s at 94°C, 30 s at 55°C, and 1 min at 72°C, and finally a 10-min incubation at 72°C. The PCR primers were as follows: 5'-TGGTGCAGTCCCTCGTCCAACC-3' and 5'-GTTCCATTGGAACTCCAAACGT-3' for SPR2 amplification, and 5'-ATGAAGATTAAGGTCGTGGCA-3' and 5'-TCCGAGTTTGAAGAGGCTAC-3' for ACT8 amplification. After separation on agarose gel, the PCR products were blotted on membranes and detected by Southern hybridization using an Alkphos Direct Labeling and Detection System (Amersham Biosciences, Piscataway, NJ).
Binary vectors pBI-SPR2, pBI-35S-SPR2HH, and pBI-SPR2-GFP were used to transform Agrobacterium tumefaciens strain MP90 and then introduced into Arabidopsis plants by floral dipping. T1 plants were selected for kanamycin resistance, and the integration of the transgenes into the plant genome was confirmed by PCR. T2 and T3 plants homozygous for the transgene were used for analysis.
Suspension cultured tobacco (Nicotiana tabacum) BY-2 cells were maintained at 27°C in a Murashige and Skoog medium containing 3% Suc, 0.2 mg/mL of KH2PO4, 0.1 mg/mL of myoinositol, 1.0 mg/L of thiamine, and 0.9 µM 2,4-dichlorophenoxyacetic acid. The binary vector pBI-SPR2-GFP was introduced into tobacco BY-2 cells using A. tumefaciens strain LBA4404, essentially as described (An, 1987
The bacterial expression vector pET-SPR2 was introduced into Escherichia coli strain BL21(DE3) Codon-Plus (Novagen). Bacteria were grown in Luria-Bertani medium supplemented with 100 mg/L of ampicillin with vigorous shaking at 37°C. When the optical density of the culture at 600 nm reached 0.4, isopropylthio-
Tubulins were prepared from cultured tobacco BY-2 cells as described (Igarashi et al., 2000
The 6x-His-tagged truncated SPR2 (amino acids 1–438) was overexpressed in E. coli strain BL21 (DE3) containing pET-SPR2N, purified through an Ni-NTA agarose column (Qiagen) and a Q-Sepharose Fast Flow column (Amersham Biosciences), and used to produce polyclonal mouse and rabbit antisera.
Microsomal and soluble proteins were prepared from Arabidopsis seedlings grown in a liquid medium as described (Nakajima et al., 2004 Immunoblot analysis of proteins prepared from tobacco BY-2 cells was done using an anti-SPR2 rabbit antiserum at a 1:1,000 dilution.
MAP fractions were prepared from tobacco BY-2 cells as described (Igarashi et al., 2000 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 permissions will be the responsibility of the requester.
After this work was submitted, molecular cloning of TOR1, which is allelic to SPR2, was published (Buschmann H, Fabri CO, Hauptmann M, Hutzler P, Laux T, Lloyd CW, Schäffner AR [2004] Helical growth of the Arabidopsis mutant tortifolia1 reveals a plant-specific microtubule-associated protein. Curr Biol 14: 1515–1521).
We thank D.H. Young and Y. Niwa for providing RH-4032 and pTH-2, respectively, and M. Yoshimura, T. Yasuhara, and M. Kuwano for technical assistance. We would like to acknowledge the Arabidopsis Biological Resource Center for providing a T-DNA knockout allele, convoluta mutant, and DNA materials. Received August 17, 2004; returned for revision September 8, 2004; accepted September 8, 2004.
1 This work was supported in part by the Ministry of Education, Culture, Sports, Science and Technology (grant nos. 15031219 to T.H. and 15770029 to T.S.), and by the Sasakawa Scientific Research Grant from the Japan Science Society (to T.S.).  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.051748. * Corresponding author; e-mail hasimoto{at}bs.naist.jp; fax 81–743–72–5529.
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