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First published online April 14, 2004; 10.1105/tpc.017830 © 2004 American Society of Plant Biologists SPIRAL1 Encodes a Plant-Specific Microtubule-Localized Protein Required for Directional Control of Rapidly Expanding Arabidopsis CellsGraduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan 1 To whom correspondence should be addressed. E-mail hasimoto{at}bs.naist.jp; fax 81-743-72-5529.
Highly organized interphase cortical microtubule (MT) arrays are essential for anisotropic growth of plant cells, yet little is known about the molecular mechanisms that establish and maintain the order of these arrays. The Arabidopsis thaliana spiral1 (spr1) mutant shows right-handed helical growth in roots and etiolated hypocotyls. Characterization of the mutant phenotypes suggested that SPR1 may control anisotropic cell expansion through MT-dependent processes. SPR1 was identified by map-based cloning and found to encode a small protein with unknown function. Proteins homologous to SPR1 occur specifically and ubiquitously in plants. Genetic complementation with green fluorescent protein fusion proteins indicated that the SPR1 protein colocalizes with cortical MTs and that both MT localization and cell expansion control are conferred by the conserved N- and C-terminal regions. Strong SPR1 expression was found in tissues undergoing rapid cell elongation. Plants overexpressing SPR1 showed enhanced resistance to an MT drug and increased hypocotyl elongation. These observations suggest that SPR1 is a plant-specific MT-localized protein required for the maintenance of growth anisotropy in rapidly elongating cells.
Directional cell expansion is fundamental to plant morphogenesis. In cells undergoing diffuse growth, directional cell elongation (anisotropic cell expansion) requires both turnover and reorganization of the cell wall–constituting polysaccharides, such as cellulose, hemicelluloses, and pectins (Brett and Waldron, 1996  ). Among them, bundles of cellulose polymers (cellulose microfibrils) appear to act as the major load-bearing polymer that specifies growth direction by preventing turgor pressure–driven wall yielding parallel to their alignment while permitting it in the opposite direction (Baskin, 2001).
It has been proposed that oriented deposition of cellulose microfibrils is controlled by cortical microtubules (MTs). Cortical MTs and cellulose microfibrils are often found to run in parallel (Baskin, 2001
Cortical MTs comprise a specialized cytoskeletal array found particularly in walled plant cells. Over the past decade, time-lapse imaging and photobleaching experiments of cortical MTs in living plant cells has increased our understanding of their dynamic properties (Wasteneys et al., 1993
The Arabidopsis spiral1 (spr1) mutant was isolated because of its characteristic helical root growth (Furutani et al., 2000
Cortical MT orientation in spr1 plants is abnormal. In root epidermal cells, cortical MTs are oriented obliquely to form left-handed helices, whereas in the ground tissue of etiolated hypocotyls, a mixture of transverse, oblique, and longitudinal arrays are observed (Furutani et al., 2000 ). The helical growth phenotype is suppressed by low concentrations of MT drugs, oryzalin, propyzamide, and taxol. When used at higher concentrations, these drugs were shown to depolymerize (oryzalin and propyzaminde) or bundle (taxol) MTs, thereby inducing radial expansion of plant cells. The spr1 phenotype is also enhanced at low temperatures, which are known to destabilize MT polymers in some plant cell types. From these observations, we hypothesized that the SPR1 protein is required for the functional integrity of cortical MTs and therefore is essential for anisotropic cell expansion (Furutani et al., 2000). In this study, we identified the SPR1 gene by a map-based cloning strategy. We determined that SPR1 encodes a small protein with unknown biochemical functions. Proteins homologous to SPR1 are found to occur ubiquitously in plants, but no related sequence was found outside the plant kingdom. Consistent with our hypothesis on the SPR1 functions, SPR1 protein was shown to associate with cortical MTs. Expression analysis and overexpression experiments suggested that SPR1 is required to maintain growth anisotropy in cells undergoing rapid expansion.
Map-Based Cloning of SPR1 Using the spr1-1 mutant allele, SPR1 was initially mapped close to the cleaved amplified polymorphisms (CAPS) marker m246 on chromosome 2 (Figure 2A). Because sequence information was not available at the time, P1 and cosmid contigs were constructed for the mapped locus. New CAPS markers were created based on the partial DNA sequences obtained from the clones in the contig and used to restrict the SPR1 gene to a 37-kb region (Figure 2A). Because spr1-1 was derived from a seed pool irradiated with fast neutrons, we searched for a deletion or a rearrangement in the 37-kb region of the spr1-1 genome. Restriction fragment length polymorphism analysis and PCR amplification of the spr1-1 and Landsberg erecta (Ler) genomic DNA within the target region revealed a 0.6-kb deletion in a 5.5-kb HindIII fragment. Transformation of spr1-1 mutants with the full-length Ler fragment rescued the helical growth defects (data not shown), indicating that the SPR1 gene lay in this region. Sequence analysis revealed a 632-bp deletion in the spr1-1 allele that disrupted two genes, At2g03670 and At2g03680 (Figure 2B), both of which were predicted to be expressed based on the presence of ESTs.
To identify which of the two genes was responsible for the spr1 phenotype, four more mutant alleles (spr1-2 through spr1-5; all proved to be allelic by noncomplementation) were sequenced. spr1-4 and spr1-5 contained deletions similar to that in the spr1-1 (Figure 2B). spr1-2 harbored a T-DNA insertion immediately downstream of the putative TATA box element of At2g03680 (Figure 2B). spr1-3 had a 10-bp deletion at an intron/exon junction of At2g03680 (Figure 2B). Furthermore, a 2.1-kb genomic fragment that spanned the entire coding region and the 0.4-kb 5' region of At2g03680, but only a small C-terminal region of At2g03670 (Figure 2B), rescued the spr1-1 mutant (Figures 1E and 1L). Taken together, we concluded that At2g03680 encodes the SPR1 gene. Consistent with large deletions or an insertion in the 5' region, spr1-1, spr1-2, spr1-4, and spr1-5 mutants did not accumulate SPR1 transcripts (Figure 3A). By contrast, spr1-3 had reduced SPR1 transcript level but slightly larger transcript size than that generated in the wild-type plants (Figure 3A). Sequencing of RT-PCR products revealed that the second intron of SPR1 was not spliced in the spr1-3 mutant. This is consistent with the 10-bp deletion in the spr1-3 allele destroying the consensus acceptor sequence of the SPR1 second intron (Figure 2B).
SPR1 Encodes a Novel Plant-Specific Protein The predicted SPR1 polypeptide consists of 119 amino acids (Figure 2C). A TBLASTN search of the GenBank nucleotide database revealed no homologous gene with known biological or biochemical functions. Numerous EST sequences, however, were found to encode polypeptides homologous to SPR1. These SPR1-related genes appear to exist ubiquitously in plants: homologs were found in all 18 plant species for which >10,000 EST sequences have been deposited (http://www.plantgdb.org). SPR1-related sequences also were found in a moss EST database (http://moss.nibb.ac.jp/). Alignments of the protein sequences deduced from some of the EST clones are shown in Figures 2C and 2D. Only the proteins from two Brassicaceous species, Thellungiella halophila and Descurainia sophia, show amino acid identity with SPR1 along the entire polypeptide length (Figure 2C). By contrast, the closest homologs from each of the non-Brassicaceous species show sequence identity only in N- and C-terminal regions, whereas the internal regions are highly variable (Figure 2D). The Arabidopsis genome contains five SPR1 homologs that we refer to as SPIRAL1-LIKE1 (SP1L1) through SP1L5. Again, these SP1L proteins share high sequence identity only in the N- and C-terminal regions (Figure 2E). The N- and C-terminal regions show limited sequence similarity to each other (Figure 2C, arrows). Based on the EST sequences deposited in the database, all SP1L genes, except for SP1L1, are predicted to be transcribed.
SPR1 Protein Is Colocalized with Cortical MTs
To confirm the MT localization of SPR1 in wild-type plants, an antiserum was produced with recombinant SPR1 proteins. On a protein gel blot, this antiserum recognized a protein band at expected size for SPR1 (12 kD) mainly in a microsomal fraction of wild-type Columbia (Col) but not in spr1-2 (Figure 4G, arrow). Because the antiserum gave many nonspecific bands in both Col and spr1-2 extracts, affinity purification was performed to give a single major band on a protein gel blot (Figure 4H, arrow). This antibody preparation was then used for immunofluoresent staining on dark-grown hypocotyls of wild-type Col seedlings. A CLSM observation of cortical cell surface revealed fibrous structures characteristic of cortical MTs (Figure 4I). In a negative control experiment, spr1-2 mutant seedlings showed only negligible fluorescence (Figures 4J). Similar fibrous structures were visualized by a CSLM observation of GFP:  -tubulin transgenic plants (Figure 4K) (Ueda et al., 1999), indicating that the SPR1 antibody stained the MTs. The observed colocalization of SPR1 with cortical MTs is consistent with the proposed role of SPR1 in MT-dependent processes (Furutani et al., 2000).
N- and C-Terminal Regions of SPR1 Are Sufficient for MT Localization
As shown in Figure 5E, replacing the internal sequence of SPR1 with GFP (P35S:NGC; N, G, and C stand for SPR1 N-region, GFP, and SPR1 C-region, respectively) partially rescued the root skewing phenotype of spr1-3. Microscopic analysis revealed that the helical growth of root epidermal cell files was also partially rescued (data not shown). By contrast, GFP fused with either the N- or C-terminal region alone (P35S:NG or P35S:GC) or free GFP (P35S:G) failed to rescue the spr1-3 defects (Table 1, Figures 5C, 5D, and 5F). Transgenic plants were then examined by CLSM to observe the distribution of the GFP fusion proteins. Only the NGC fusion protein was localized to cortical MTs (Figure 5I), whereas the NG, GC, and G proteins were all distributed throughout the peripheral cytoplasm (Figures 5G, 5H, and 5J). These results indicate that the conserved N- and C-terminal regions act together in targeting SPR1 proteins to cortical MTs and that the internal region may play minor roles as compared with the conserved N- and C-terminal regions. The coincidence between phenotypic rescue and MT localization further suggests that the primary function of SPR1 is related to its association with cortical MTs.
Expression Patterns of SPR1
In young seedlings, the expression of SPR1 appeared to be dependent on light and developmental stage. When seedlings were grown under continuous illumination of white light, hypocotyls showed negligible staining, whereas cotyledons started to express GUS after 4 d of growth (Figure 6D). By contrast, when seedlings were grown in the dark, hypocotyls started to express GUS after 3 to 4 d of growth, whereas cotyledons remained unstained (Figures 6G to 6K). The light-dependent induction of GUS expression in the cotyledons was confirmed by exposing dark-grown seedlings to continuous light. One day after the transfer, cotyledons of the light-exposed seedlings started to express GUS, whereas those of control seedlings left in the darkness remained unstained (cf. Figures 6E and 6F). GUS staining in the root was constitutive; no difference was observed between light- and dark-grown seedlings (data not shown). Interestingly, the region of strong GUS stain moved acropetally along the hypocotyls of dark-grown seedlings. Three- or 3.5-day-old seedlings (corresponding to 24 to 36 h after germination) showed GUS stain preferentially at the basal part of hypocotyls (Figures 6G and 6H). Four- or five-day-old seedlings were stained in the middle region of hypocotyls (Figures 6I and 6J). In 6-d-old seedlings, GUS stain revealed expression only in the apical region of fully elongated hypocotyls (Figure 6K). A similar expression pattern was detected by immunostaining of dark-grown wild-type hypocotyls (see supplemental data online).
In summary, high SPR1 expression was associated with tissues undergoing rapid cell expansion, including the root elongation zone, hypocotyls of dark grown-seedlings, and cotyledons of light-grown seedlings (Schiefelbein and Benfey, 1991
Overexpression of SPR1
The P35S:SPR1 plants were found to be moderately more resistant to long-term treatment with the MT-depolymerizing drug propyzamide. When grown on a medium containing 5 µM propyzamide, plants from two independent P35S:SPR1 lines (1 and 2) showed marginal survival, whereas both Ler and spr1-1 plants died (Figure 7B).
To investigate the role of SPR1 in cell elongation, we compared the cell elongation kinetics of P35S:SPR1, wild-type Col, and spr1-2 plants using dark-grown hypocotyls, the extension of which relies solely on cell expansion but not on cell division (Gendreau et al., 1997
SPR1 Encodes a Plant-Specific MT-Localizing Protein Our map-based cloning lead to the identification of SPR1 as a novel protein composed of 119 amino acid residues. In the nucleotide database, the SPR1 cDNA sequence is annotated as a nitrilase-associated protein, based on an interaction in the yeast two-hybrid system (GenBank accession number Z96936; D. Bartling, personal communication). Our yeast two-hybrid assay, however, could not reproduce this interaction (R. Prieto and T. Hashimoto, unpublished results). The deposited sequence has a single base pair deletion close to the SPR1 stop codon. If this is not a simple sequencing error but has in fact been incorporated accidentally during the cloning procedure, the translated protein would have 27 unrelated amino acids translated from the 3' noncoding region. Such an artificial sequence might account for the reported interaction with nitrilase in yeast. Under these circumstances, we will solely use SPR1 as the gene symbol. The SPR1 amino acid sequence did not show homology to known proteins with defined functions, nor did it contain a structural motif indicative of its activity. Nevertheless, a database search revealed that SPR1-related proteins occur ubiquitously in plants; many monocot and dicot species as well as a moss species possess multiple SPR1-related genes. The deduced protein sequences from those ESTs and SPR1 are similar only in N- and C-terminal regions, except for two from other species in the Brassiceae, and therefore are closely related to Arabidopsis. The internal regions are highly variable but are similar in length. This suggests that amino acid residues important for the SPR1 function(s) reside mainly in the N- and C-terminal regions, whereas the internal region functions as a spacer that holds the two conserved polypeptides in a defined distance. The results of our GFP fusion protein expression study shown in Figure 5 support this hypothesis. Only GFP fused with the SPR1 polypeptides at both N and C termini could partially rescue the spr1 defect. The incomplete rescue may be attributable to either nonoptimal spacing by the GFP polypeptide, imperfect excision of the SPR1 N- and C-terminal domains, conformational constraint at the fusion points, or a combination of these problems. Use of the 35S promoter should not be a problem because intact SPR1 proteins expressed under P35S could completely rescue the spr1 defects (data not shown). Taken together, these findings suggest that SPR1 and its homologs have a similar function, even though their internal regions are highly divergent.
Observation of PSPR1:SPR1:GFP transgenic plants and immunostaining revealed that SPR1 colocalizes with cortical MTs in plant cells. Previously, we hypothesized that SPR1 is involved in MT-dependent processes based on the observation of cortical MTs and growth phenotype under the conditions that either stabilize or destabilize MTs (Furutani et al., 2000
Highly ordered cortical MT arrays are a plant-specific cytoskeletal structure that play essential roles in directed expansion of plant cells (Burk et al., 2001
Role of SPR1 in Cell Elongation
Second, SPR1 is expressed preferentially in tissues with rapid cell elongation. In light-grown seedlings, SPR1 is expressed in cotyledons but not in hypocotyls. By contrast, in dark-grown seedlings, SPR1 is expressed in hypocotyls but not in cotyledons. The opposite effect of light on the expression of SPR1 in hypocotyls and cotyledons suggests that light is not a simple switch for the SPR1 expression. Rather, SPR1 expression seems to be controlled as a part of a light-dependent developmental program. In the light, plants undergo photomorphogenesis in which cotyledons rapidly expand, whereas hypocotyls show only limited elongation. In the dark, plants adopt skotomorphogenesis in which hypocotyls rapidly elongate, whereas cotyledons do not change their size (Neff and Van Volkenburgh, 1994 Third, transgenic plants overexpressing SPR1 showed a small but reproducible increase in the elongation of dark-grown hypocotyls. The relatively small increase suggests that the wild-type level of SPR1 is already close to the amount required for maximum elongation or that SPR1 can accelerate ongoing cell elongation but cannot trigger new cell elongation. The same experiment also showed reduced hypocotyl length of spr1 mutants. The measurements of spr1 hypocotyls, however, should be interpreted with caution. Hypocotyl elongation of spr1 started to deviate from that of wild-type plants only 3 d after germination. This is exactly the time when upper hypocotyls of spr1 mutant started to show helical growth, thereby reducing the overall length of hypocotyls. Therefore, the role of SPR1 in hypocotyl elongation should be deduced only from gain-of-function experiments such as overexpression.
Origin of the Helical Growth Phenotype If helical growth is a manifestation of partially impaired anisotropic cell expansion, then why do null spr1 alleles show helical growth rather than isotropic cell expansion? A trivial possibility would be that SPR1 does not actively participate in maintaining anisotropic cell expansion. However, given the fact that SPR1 functions through conserved N- and C-terminal regions, it is more plausible that SPR1 and SP1Ls share partially redundant functions and that the null spr1 mutation represents partial loss of the SPR1/SP1L gene family. Our preliminary characterization of double mutants of SPR1 and some SP1Ls suggests that this is the case (K. Nakajima, T. Kawamura, and T. Hashimoto, unpublished results).
We have previously proposed a model in which the helical growth phenotype of spr1 arises from differential effects of the spr1 mutation on epidermis and ground tissue (i.e., relatively strong radial expansion of inner ground tissue causes the outer and inner cells to have different longitudinal length). To compensate for the different cell length, epidermal cells become inevitably skewed (Furutani et al., 2000
Plant Materials and Growth Conditions Isolation of spr1-1 (Ler), spr1-2 (Col), spr1-3 (Col), and spr1-4 (Wassilewskija ecotype) mutant alleles have been described (Furutani et al., 2000 ). spr1-5 (Wassilewskija ecotype) was isolated from the Institut National de la Recherche Agronomique T-DNA population (http://flagdb-genoplante-info.infobiogen.fr/projects/fst/DocsIntro/introCollection.html). Plants were grown as described previously (Furutani et al., 2000), unless noted otherwise.
Map-Based Cloning Genomic DNAs from spr1-1 and Ler were digested with HindIII and subjected to a genomic DNA gel blot using each of the two cosmid clones spanning the 37-kb segment as a probe. A restriction fragment length polymorphism was found in a 5.4-kb Ler fragment, which in spr1-1 was 4.7 kb. A series of PCR amplification steps were performed to scan the 37-kb region and successfully detected 0.6-kb deletion within the 5.4-kb HindIII fragment. This fragment was introduced into the spr1-1 mutant using pBIN19 binary vector and shown to rescue the spr1 mutant defects. Subsequent sequencing of five spr1 alleles predicted that the gene At2g03680 encodes SPR1. This was further confirmed by genetic complementation of spr1-1 with a 2.1-kb SpeI-SnaBI genomic fragment that spanned the 0.4-kb upstream region relative to the putative TATA box, all exons and introns, and the 0.6-kb downstream region of At2g03680.
RNA Gel Blot Analysis
Plasmid Construction, Plant Transformation, and Protein Production in Escherichia coli
For plant transformation, each DNA construct was transferred to either the pBIN19 (Frisch et al., 1995 Recombinant protein was produced in the E. coli strain BL21. All proteins were expressed as a soluble form at 37°C. Proteins were extracted by sonication and affinity purified using amylose resin (New England Biolabs) for MBP:SPR1 or nickel-nitrilotriacetic acid agarose (Qiagen) for TRX:SPR1 according to the manufacturers' instructions. The eluted proteins were further purified by anion-exchange chromatography using either a Mono Q fast-protein liquid chromatography column or Q-Sepharose fast flow resin (Amersham Biosciences).
Immunological Technique For plant protein extraction, Arabidopsis seedlings were sliced into small pieces by razor blades and ground in ice-cold extraction buffer (50 mM Hepes-KOH, pH 7.5, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM DTT, 0.25 M sucrose, 20 µg/mL of pepstatin A, and 20 µg/mL of leupeptin) containing 0.1 mg/mL of butylated hydroxytoluene and 10% (v/v) polyclar AT. After filtration through two layers of Miracloth (Calbiochem, San Diego, CA), the insoluble debris was removed by centrifugation at 7000g for 15 min at 4°C. Microsomal proteins then were separated from the supernatant by centrifuging the protein extracts twice at 50,000g for 30 min at 4°C. Immunoblot analysis was performed with Hybond-P membrane and the ECL-plus kit (Amersham Biosciences) according to the manufacturer's instructions.
Whole mount immunostaining was done by the method described for MT labeling (Sugimoto et al., 2000
Microscopy
Histochemical GUS Staining Sequence data from this article have been deposited with the EMBL/GenBank data libraries under the following accession numbers: Arabidopsis SPR1, AY464947; Arabidopsis SPR1 homologs, SP1L1, NM_102400 (At1g26360); SP1L2, NM_105590 (At1g69230); SP1L3, NM_111085 (At3g02180); SP1L4, NM_121564 (At5g15600); SP1L5, NM_118480 (At4g23496). SPR1 homologs from other species are T. halophila, BM985496; D. sophia, BG321626; Soybean, BI787806; Tomato, AW219464; Cotton, BE052696; Maize, BI431085; and Rice, CA758698. The moss SPR1 homolog sequence (contig 465; Physcomitrella patens) was retrieved from the Physcobase database (http://moss.nibb.ac.jp). Nucleotide sequence for SP1L1 is very likely to be misannotated in the database, where coding regions for SP1L1 and the preceding hydrolase-like gene are fused together. In this study, we separated the deposited nucleotide sequence into two genes, and the latter half was designated SP1L1.
We thank J. Schiefelbein for the spr1-2 mutant, K. Nakamura and A. Morikami for the spr1-3 mutant, A. Arnim for GFP plasmids, and Tomomi Kawamura for technical assistance. We are grateful to G.O. Wasteneys for valuable comments on the manuscript and T. Fujita for introducing the moss database. P1 and cosmid genomic libraries were provided by Mitsui Gyosai Bio. Some seeds were obtained through ABRC. This work was supported in part by grants from the Ministry of Education, Science, Sports, and Culture and from Japan Atomic Energy Research Institute to T.H.
Online version contains Web-only data.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Takashi Hashimoto (hasimoto{at}bs.naist.jp). Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.017830. Received September 30, 2003; accepted March 2, 2004.
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