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Plant Cell. May 2005; 17(5): 1387–1396.
PMCID: PMC1091762

Crown rootless1, Which Is Essential for Crown Root Formation in Rice, Is a Target of an AUXIN RESPONSE FACTOR in Auxin SignalingW in Box

Abstract

Although the importance of auxin in root development is well known, the molecular mechanisms involved are still unknown. We characterized a rice (Oryza sativa) mutant defective in crown root formation, crown rootless1 (crl1). The crl1 mutant showed additional auxin-related abnormal phenotypic traits in the roots, such as decreased lateral root number, auxin insensitivity in lateral root formation, and impaired root gravitropism, whereas no abnormal phenotypic traits were observed in aboveground organs. Expression of Crl1, which encodes a member of the plant-specific ASYMMETRIC LEAVES2/LATERAL ORGAN BOUNDARIES protein family, was localized in tissues where crown and lateral roots are initiated and overlapped with β-glucuronidase staining controlled by the DR5 promoter. Exogenous auxin treatment induced Crl1 expression without de novo protein biosynthesis, and this induction required the degradation of AUXIN/INDOLE-3-ACETIC ACID proteins. Crl1 contains two putative auxin response elements (AuxREs) in its promoter region. The proximal AuxRE specifically interacted with a rice AUXIN RESPONSE FACTOR (ARF) and acted as a cis-motif for Crl1 expression. We conclude that Crl1 encodes a positive regulator for crown and lateral root formation and that its expression is directly regulated by an ARF in the auxin signaling pathway.

INTRODUCTION

The plant root system consists of various components, including seminal, adventitious, and lateral roots. While the seminal root develops during embryogenesis, adventitious and lateral roots develop from differentiated cells postembryonically. Root architecture is a key determinant of nutrient and water use efficiency in crops. In contrast with Arabidopsis thaliana, in which adventitious roots are rarely formed, monocot plants produce numerous crown roots, a kind of adventitious root that is dominant in the root system of cereals. Many mutants that affect root development have been identified and characterized in Arabidopsis, contributing to our understanding of the genetic mechanisms of root development (Casimiro et al., 2003; Casson and Lindsey, 2003; Schiefelbein, 2003). However, only a small number of mutants have been found in monocots, and no genes corresponding to these mutants have been identified.

The endogenous phytohormone auxin is essential for root development. Exogenous treatment with auxin induces the ectopic formation of lateral and adventitious roots, although the optimal auxin concentration for inducing ectopic formation differs between lateral and adventitious roots (Schiefelbein, 2003). It has also been reported that genes involved in the auxin signaling pathway function in root development. Gain-of-function mutants of Arabidopsis AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) genes, which encode negative regulators of auxin signaling, produce phenotypes with reduced or no lateral roots (Reed, 2001; Marchant et al., 2002). AUX/IAA proteins regulate gene expression by interacting with AUXIN RESPONSE FACTOR (ARF) proteins, which function as positive regulators of auxin signaling (Reed, 2001). The loss-of-function mutant of Arabidopsis ARF8 showed increased lateral root formation, and overexpression of ARF8 in transgenic Arabidopsis inhibited lateral root formation (Tian et al., 2004). Based on this evidence, the important role that auxin plays in root development is apparent; however, the targets and molecular mechanisms downstream of AUX/IAA and ARF proteins in root development are still unresolved.

To study the mechanism of root formation, we previously isolated a rice crown rootless1 (crl1) mutant that was defective in the formation of crown roots (Inukai et al., 2001). Because crl1 shows severe defects in crown root formation as well as a reduced number of lateral roots on seminal roots, we suspected that Crl1 is involved in auxin-related root formation. In this work, we isolated the Crl1 gene and characterized its biological function in terms of auxin signaling. Crl1 encodes an ASYMMETRIC LEAVES2 (AS2)/LATERAL ORGAN BOUNDARIES (LOB) domain transcription factor, which has not previously been reported as modulating root development. We also showed that auxin and an AUX/IAA protein tightly regulate Crl1 expression and that Crl1 is a direct target of an ARF protein in rice (Oryza sativa). Thus, these findings have important implications for our understanding of the function of auxin in root development.

RESULTS

Characterization of the crl1 Mutant

Two-week-old crl1 mutants had normal seminal root development but did not form any crown roots, whereas wild-type plants formed several crown roots at the same developmental stage (Figures 1A and 1B). From serial cross sections, we observed the internal structure of the node-containing stem regions where crown root formation occurs. In the wild type, crown root primordia (arrow in Figure 1C) formed on the outside, adjacent to the peripheral vascular cylinder (arrowhead in Figure 1C) of the stem. By contrast, crl1 did not produce any crown root primordia (Figure 1D). The number of lateral roots from a seminal root in crl1 also decreased to ~70% of that in the wild type (Figures 1A and 1B), indicating that Crl1 is involved in both crown root and lateral root formation. At later stages of development, crl1 occasionally produced crown roots (Figures 1E and 1F). Given adequate supplies of water and fertilizer, the aboveground tissues and organs of crl1 grew normally and did not differ significantly from the wild type morphologically, even at harvest (Figures 1G and 1H). This indicates that Crl1 is specifically involved in root formation but not in shoot development.

Figure 1.
Phenotypes of crl1.

When wild-type seedlings were treated with IAA, 2,4-D, and naphtalene acetic acid (NAA), the numbers of both crown and lateral roots increased in a dose-responsive manner up to 10−6 M of IAA or 10−9 M of 2,4-D, with inhibition at higher concentrations (Tables 1 and and2).2). Conversely, the stimulatory effect of exogenous auxin was not observed in crl1, whereas its inhibitory effect was seen in lateral root formation at 10−7 M 2,4-D and 10−6 M NAA treatment (Table 2). These results indicate that the positive effect of auxin on crown and lateral root formation is defective in crl1.

Table 1.
Effect of Exogenous Auxin Treatment on Crown Root Formationa
Table 2.
Effect of Exogenous Auxin Treatment on Lateral Root Formationa

We also examined the root gravitropic response in crl1 by measuring the curvature after gravistimulation at 90° to the vertical. Wild-type roots responded sharply to the change in the gravity vector, whereas the response of crl1 roots was impaired (Figure 2A). The root tip angle of wild-type and crl1 roots was compared (θ in Figure 2B). Approximately 80% of wild-type roots had root tip angles of 61 to 80°, and no plants had an angle of <40°; by contrast, 60% of crl1 roots had angles of <40° (Figure 2C). Therefore, the gravitropic response was also impaired in crl1.

Figure 2.
Gravitropic Phenotype in Seminal Roots of crl1.

Crl1 Encodes an AS2/LOB-Domain Protein

The Crl1 locus was cloned using the map-based chromosome walking procedure. The Crl1 locus was mapped on the short arm of chromosome 3, between two markers ~6 kb apart (Figure 3A). This region includes two predicted open reading frames (ORFs). By comparing the nucleotide sequences of crl1 and the wild type, we concluded that Crl1 has an ORF that encodes a protein of 259 amino acid residues in two exons (Figure 4A). There was a single nucleotide substitution in crl1 that produced a single amino acid change, whereas no mutation was detected in the other ORF. Complementation analysis based on introduction of a 4.6-kb genomic DNA fragment containing the entire candidate gene confirmed that the crl1 phenotype is caused by a loss-of-function mutation in this predicted Crl1 gene (Figure 3B).

Figure 3.
Map-Based Cloning and Phenotypic Complementation by the Introduction of Crl1.
Figure 4.
Structure of Crl1.

The amino acid sequence deduced from the cDNA sequence revealed that Crl1 encodes a protein containing an AS2/LOB domain (Figure 4B). The recently identified AS2/LOB protein family includes a conserved domain with ~100 amino acid residues (Iwakawa et al., 2002; Shuai et al., 2002). The AS2/LOB domain of Crl1 shared the conserved amino acid residues of Arabidopsis AS2/LOB proteins (i.e., CX2CX6CX3C at the N terminus [C-motif] and a GAS region at the C terminus [GAS-motif]). We performed a BLAST search using the AS2/LOB domain sequence of Crl1 and found that some AS2/LOB proteins in Arabidopsis, maize (Zea mays), and rice are more similar to Crl1 than to other AS2/LOB proteins (Figure 4C). It is interesting that AS2-LIKE18/LOB DOMAIN16 (ASL18/LBD16) and ASL16/LBD29, which are classified in the same group with Crl1, are specifically expressed in the roots of Arabidopsis (Shuai et al., 2002), suggesting that the members in this group are involved in root formation or development, as is the case with Crl1.

Analysis of Crl1 Expression

We examined the expression of Crl1 in various organs. Because no band was detected on RNA gel blots, we performed semiquantitative RT-PCR analysis to estimate Crl1 transcript levels (Figure 5A). High levels of Crl1 transcripts accumulated in unelongating basal internodes. Crl1 was moderately expressed in roots and flowers but not in other organs. In rice, root formation occurs not only in basal internodes and roots but also at the base of spikelets (flowers of rice) when grown under stress conditions (Takeoka and Shimizu, 1974); consequently, all organs expressing Crl1 have the ability to form roots, whereas organs without Crl1 expression do not.

Figure 5.
Expression Pattern of Crl1.

We also examined the Crl1 expression pattern indicated by β-glucuronidase (GUS) activity under the control of the Crl1 promoter (Figures 5B to 5L). In shoots, GUS staining was observed only at the base of shoots (Figures 5B and 5C). In this region, the staining was preferentially localized in parenchyma cells adjacent to the peripheral vascular cylinder of the stem, where crown root primordia were formed (Figures 5D and 5E). In the seminal and crown roots, spots of GUS staining were observed (Figure 5F). The GUS staining was preferentially localized in tissues from which lateral roots are initiated (Figures 5K and 5L). In rice roots, lateral root primordia are initiated by anticlinal cell divisions, followed by periclinal cell divisions, in the pericycle and endodermis (Kawata and Shibayama, 1965; Figures 5I and 5J). In the initial stage of primordium formation, GUS staining was observed in the pericycle cells (Figures 5I and 5J), and then it gradually disappeared in the developed primordium (Figures 5G and 5H). The localized expression of Crl1 in stems and roots corresponds well to the areas of crown and lateral root initiation, confirming that Crl1 is involved in the initiation of these root systems.

Auxin Signaling Regulates Crl1 Expression

We examined the effect of auxin on Crl1 expression using semiquantitative RT-PCR. The expression of Crl1 was induced within 1 h and increased dramatically until 3 h after IAA treatment, after which it gradually decreased (Figure 6A). We also observed the expression of OsIAA4, which is a member of the early auxin response AUX/IAA family in rice (I. Umemura and M. Matsuoka, unpublished results). The induction profile of OsIAA4 was similar to that of Crl1 (Figure 6A). The early auxin response of Crl1 expression suggests that Crl1 is a member of the early auxin response family. Therefore, we examined the effect of the protein synthesis inhibitor cycloheximide (CHX) on the auxin-dependent induction of Crl1 and OsIAA4 (Figure 6B). As expected, the auxin-dependent induction of OsIAA4 was not inhibited by CHX, and slight expression was induced by CHX alone, as with Arabidopsis AUX/IAA genes (Abel et al., 1995). A similar induction profile was observed in the case of Crl1, strongly suggesting that de novo protein synthesis is not required for Crl1 induction by auxin.

Figure 6.
Auxin Induces Crl1 Expression.

We also compared the expression of GUS under the control of the Crl1 and DR5 promoters (Figures 6C and 6D). DR5 is often used as an artificial promoter responding to auxin signals in Arabidopsis (Sabatini et al., 1999). This promoter was also used as a marker to visualize the in vivo distribution of auxin in rice (Scarpella et al., 2003). The GUS staining controlled by the DR5 promoter was broadly observed in the outer parenchyma cells of the peripheral vascular cylinder in the stem (Figure 6D), including where Crl1 was expressed (Figures 5D and and6C).6C). This confirms that the Crl1 expression colocalizes with auxin distribution in vivo.

Recent studies have revealed that auxin signaling is regulated by the auxin-stimulated degradation of a family of negative regulators called AUX/IAA proteins (Gray et al., 2001; Tiwari et al., 2001; Zenser et al., 2001). These negative regulator proteins interact with another large family of plant-specific transcription factors called ARF proteins, which are positive transcriptional regulators in auxin signaling (Hagen and Guilfoyle, 2002). Because RT-PCR and GUS analyses indicated that the expression of Crl1 is under control of auxin (Figures 6A to 6D), we analyzed whether Crl1 expression depends on the degradation of AUX/IAA proteins.

To produce constitutively active rice AUX/IAA protein, we mutagenized the conserved Pro of OsIAA3, which is located in the degradation-related domain (domain II), to Leu (Figure 6E). Because the mutagenesis of OsIAA3 caused the constitutive suppression of auxin signaling, transformed calli rarely formed regenerated seedlings. To alleviate this problem, we used a steroid hormone-inducible system to control the suppressive function of the mutagenized OsIAA3 on treatment with the steroid hormone dexamethasone (DEX). We generated >20 transgenic plants expressing a fusion protein of OsIAA3P58L and the steroid hormone binding domain of the glucocorticoid receptor (GR). Few of the transgenic plants showed abnormalities without DEX treatment, whereas they showed diverse auxin-related abnormalities, including defects in root formation and stunted seedlings, with DEX treatment (our unpublished results). Using these transgenic plants, we examined the AUX/IAA-dependent expression of Crl1 with or without DEX treatment. We used two independent lines, which showed severely or mildly altered phenotypes when treated with DEX. As mentioned above, Crl1 expression was clearly induced by IAA treatment (cf. IAA− DEX− and IAA+ DEX− in Figure 6F). However, the induction by auxin was almost completely inhibited by DEX treatment in line 1 (the severe phenotype) and strongly inhibited in line 2 (the intermediate phenotype) (IAA+ DEX+ in Figure 6F). These results demonstrate that the degradation of AUX/IAA proteins is essential for the auxin-dependent expression of Crl1 and strongly suggest that Crl1 expression is positively regulated by ARF proteins.

ARF Protein Binds to the Auxin Response Element in the 5′ Flanking Sequences of Crl1

The auxin response element (AuxRE) containing the TGTCTC motif has been identified in the promoters of some early auxin response genes, and ARFs bind AuxRE to regulate the transcription of these genes (Hagen and Guilfoyle, 2002). Because Crl1 contains two TGTCTC motifs (AuxRE1 and AuxRE2 with its inverted sequence, GAGACA) in its promoter region (Figure 7A), we wondered whether ARFs interact with these sequences. To examine this possibility, we performed an electrophoresis mobility shift assay. The recombinant rice ARF protein (OsARF1; Waller et al., 2002) expressed in Escherichia coli bound to fragment 3 containing AuxRE2. This resulted in a band with lower mobility than that of the free fragment, whereas the other fragments, including fragment 1 containing AuxRE1, were not shifted (Figure 7B). The binding of OsARF1 with fragment 3 did not occur when one nucleotide of the AuxRE2 sequence was changed (fragment M3), demonstrating that the interaction between fragment 3 and OsARF1 depends on AuxRE2.

Figure 7.
AuxRE in the CrlI Promoter Is Essential for Interaction of OsARF1 and Expression of Crl1.

Because OsARF1 binds to AuxRE2 in vitro, this sequence may act as a cis-acting factor for transcriptional regulation of Crl1 by rice ARF proteins in vivo. To examine this possibility, we introduced the single nucleotide substitution at AuxRE2 corresponding to the fragment M3 and generated a fusion construct with the GUS reporter gene, which was transformed into wild-type rice. As mentioned previously, GUS staining was observed at the base of shoots in transformants carrying the wild-type promoter (Figure 7C). However, this staining was not observed in transformants carrying the mutant promoter (Figure 7D). Thus, AuxRE2 in the Crl1 promoter is essential for Crl1 expression.

DISCUSSION

In addition to the defect in crown root formation, crl1 showed additional auxin-related abnormal phenotypic traits in the roots, such as decreased lateral root number, auxin insensitivity in lateral root formation, and impaired root gravitropism, whereas no altered phenotypic traits were observed in aboveground organs. Consistent with these phenotypes, Crl1 expression was localized in parenchyma cells adjacent to the peripheral vascular cylinder of the stem and in the pericycle and endodermis cells of seminal and crown roots, where crown and lateral roots are initiated, respectively.

Crl1 encodes a member of the plant-specific AS2/LOB protein family. There are 43 genes for AS2/LOB proteins in the Arabidopsis genome (Shuai et al., 2002), and one of them, AS2, is required for the formation of symmetric leaves (Iwakawa et al., 2002). However, there is no information about the biochemical activity of AS2/LOB domain proteins. Recently, Zgurski et al. (2005) reported that an asymmetric auxin response precedes asymmetric cell division patterns and leaf expansion in as2. Treatment of as2 leaves with either exogenous auxin or an auxin transport inhibitor eliminates the asymmetric auxin response and the subsequent asymmetric leaf development. This result suggests that AS2 functions in regulating the symmetric auxin response in Arabidopsis leaves. As discussed below, however, we believe that Crl1 is specifically involved in AUX/IAA auxin signaling in root development and that Crl1-like AS2/LOB proteins, such as Crl1, ASL18/LBD16, and ASL16/LBD29, may belong to a new small protein family, which specifically functions in auxin-regulated root development. Consistent with this hypothesis, the expression of both ASL18/LBD16 and ASL16/LBD29 was induced by auxin, and their auxin-dependent induction was severely impaired in T-DNA insertion lines for ARF genes of Arabidopsis (Okushima et al., 2005).

The pattern of Crl1 expression was similar to the pattern of GUS expression driven by the DR5 promoter, and Crl1 expression was upregulated by auxin without the de novo synthesis of any other proteins. Moreover, the constitutive activation of an AUX/IAA protein in transgenic rice disturbed the induction of Crl1 expression by auxin treatment. Recent molecular genetic studies have demonstrated that AUX/IAAs play a central role in auxin signaling (Hagen and Guilfoyle, 2002; Liscum and Reed, 2002). AUX/IAAs function as negative regulators in auxin signaling by direct interaction to prevent the functions of ARFs (Ulmasov et al., 1997; Tiwari et al., 2001, 2003). Auxin treatment promotes the degradation of AUX/IAAs by enhancing the interaction with an SCFTIR complex to release the prevented ARF function (Gray et al., 2001). These observations and our results strongly suggest that Crl1 is a direct target of ARF. Indeed, Crl1 contains two putative AuxREs in its promoter region, and the proximal AuxRE specifically interacts with rice ARF and acts as a cis-motif for Crl1 expression in rice.

The control of AUX/IAAs by the auxin signaling pathway regulates a wide variety of plant growth and developmental processes. For example, mutations in AXR3/IAA17 result in diverse auxin-related phenotypes, including reduced root elongation, increased adventitious root formation, and a lack of root gravitropism (Leyser et al., 1996; Rouse et al., 1998). The SLR/IAA14 and IAA28 gain-of-function mutations confer severe defects in lateral root formation (Rogg et al., 2001; Fukaki et al., 2002). The AUX/IAA-controlling pathway also regulates the expression of a variety of genes. The most thoroughly studied of these are members of the AUX/IAA, GH3, and SAUR gene families, which are early auxin response genes (Hagen and Guilfoyle, 2002). Although the biological function of the SAUR genes remains unclear, AUX/IAAs function as mediators in auxin responses, as mentioned above, and GH3s function in auxin homeostasis by producing amino acid conjugates of IAA (Staswick et al., 2005). However, there has been no evidence that these early auxin response genes are directly involved in auxin-related phenotypes, such as root formation, root elongation, and root gravitropism (i.e., there has been no direct link between the AUX/IAA-controlling signal pathway and auxin-regulated phenotypes).

In this study, we demonstrated that Crl1 functions as a mediator linking the control of AUX/IAAs by auxin to the initiation of crown and lateral root development. In our model of Crl1 function in root development (Figure 8), auxin triggers the degradation of AUX/IAAs, which interact with ARFs. The released ARFs interact with the AuxRE in the Crl1 promoter to trigger its transcription in the crown and lateral root initiation areas, resulting in root initiation. Crl1 expression is insufficient to initiate crown or lateral roots because the ectopic expression of Crl1 by the constitutive rice Actin1 promoter did not induce any ectopic roots in transgenic rice (our unpublished results). This indicates that there is another factor(s) essential for crown and lateral root formation in addition to Crl1. Further studies are needed to understand how Crl1 functions and what factors mediate the signal after Crl1 to initiate root formation.

Figure 8.
Auxin Signaling Pathway Leading to Crl1 Expression in Root Formation.

METHODS

Characterization of Mutants

Seedlings of wild-type rice (Oryza sativa cv Taichung 65) and crl1 were grown in a greenhouse at 30°C under continuous light. Histological analysis was performed as previously reported (Inukai et al., 2001). To investigate the effects of auxin on root growth and development, seeds of the wild type and crl1 were sown on agar medium containing various concentrations of IAA, 2,4-D, and NAA (Sigma-Aldrich, St. Louis, MO).

Molecular Cloning, Sequence Alignment, and Phylogenetic Tree Construction

To map Crl1, linkage analysis was performed using an F2 population of ~2500 plants derived from the cross between crl1 (japonica variety) and Kasalath (indica variety). A BLAST search was performed, as previously reported (Sakamoto et al., 2004). The predicted protein sequences were initially clustered using ClustalW (Thompson et al., 1994; see Supplemental Figure 1 online). TreeView was used to generate the graphical output (Page, 1996). The numbers at the branching points indicate the percentage of times that each branch topology was found during bootstrap analysis (n = 1000).

Expression Analysis

Semiquantitative RT-PCR was performed with DNase-treated total RNA using the Omniscript reverse transcription kit (Qiagen, Valencia, CA). The PCR (30 cycles) was performed essentially as described by the manufacturer. The primer sequences were 5′-AGCAACGTGTCCAAGCTGCT-3′ and 5′-GTCCTGGTGGTGTATCCCTT-3′ for Crl1 and 5′-GGCATTCCCGGTGCCCATGA-3′ and 5′-GTCCATCGCCTATGGTGCGAC-3′ for OsIAA4. These primers specifically amplified the target gene sequences (data not shown). To examine the effect of exogenous auxin treatment on Crl1 and OsIAA4 expression, roots of wild-type or transgenic seedlings were submerged in a solution of 1 μM IAA. For inhibition of protein synthesis, seedlings were first soaked in a solution of 10 μM CHX (Sigma-Aldrich) and then incubated for 3 h in a solution containing 10 μM CHX and 1 μM IAA, or 10 μM CHX and ethanol as a control. To induce the function of constitutive active OsIAA3, roots of transgenic seedlings were submerged for 3 h in a solution of 1 μM IAA with or without 10 μM DEX (Sigma-Aldrich).

Plasmid Constructs and Plant Transformation

For complementation of the crl1 mutation, the wild-type genomic sequence from −2581 to +1969 (taking the translation initiation site as +1) was amplified by PCR and cloned into pBI121. For the Crl1 promoter-GUS construct, the wild-type genomic sequence from −2581 to +518 was amplified by PCR and introduced in front of the GUS reporter gene of pBI-Hm (kindly provided by Kenzo Nakamura, Nagoya University, Nagoya, Japan) to produce a fusion with the GUS reporter gene. The DR5 promoter-GUS construct was generated as reported previously (Scarpella et al., 2003). Nucleotide substitutions in the Crl1 promoter and OsIAA3 cDNA were introduced by PCR as previously reported (Sakamoto et al., 1999). To create the OsIAA3P58L:GR fusion protein, the stop codon of mutated OsIAA3 was replaced with a SmaI site by PCR and introduced in front of the steroid binding domain of the human GR as previously reported (Sakamoto et al., 2001). OsIAA3P58L:GR was then cloned between the rice actin1 promoter and the nopaline synthase polyadenylation signal of the hygromycin-resistant binary vector pAct-Hm. This vector is modified from pBI-Hm (Ohta et al., 1990) and contains a rice actin1 promoter. The resulting fusion construct was introduced into Agrobacterium tumefaciens strain EHA101 by electroporation. Agrobacterium-mediated transformation of rice was performed as described previously (Hiei et al., 1994). Transgenic plants were selected on media containing 50 mg L−1 hygromycin, and we analyzed 30, 20, and 25 transformants carrying Crl1, DR5, and the mutated Crl1 promoter-GUS construct, respectively.

Electrophoresis Mobility Shift Assay

To produce a recombinant OsARF1 protein, the full-length OsARF1 cDNA was inserted in the sense orientation as a translational fusion into the pET-32a expression vector (Novagen, Madison, WI) and expressed in BL21 (DE3) Escherichia coli cells (Stratagene, La Jolla, CA). The Crl1 promoter fragments (~350 bp) were labeled with [32P]dATP using the Klenow fragment and purified on Sephadex G-50 columns. DNA binding reactions were performed as previously reported (Sakamoto et al., 2001).

Sequence data for the AS2/LOB genes have been deposited with the EMBL/GenBank data libraries under the following accession numbers: ASL4/LOB, AF447897; AS2/LBD6, AF447887; ASL18/LBD16, AF447890; ASL20/LBD18, AF447891; ASL23/LBD19, AF432232; ASL3/LBD25, AF447892; ASL16/LBD29, AF447893; Crl1, AB200234; OsCrll1, AB200235; OsCrll2, AB200236; OsCrll3, AB200237; OsCrll4, AB200238; ZmCrll1, BG873644; ZmCrll2, BE050765.

Supplementary Material

[Supplemental Data]

Acknowledgments

We are grateful to Hiroko Ohmiya (Nagoya University) for technical assistance. This work was supported in part by a Grant-in-Aid from the Center of Excellence to Y.I. and M.M. and by a Grant-in-Aid from the Ministry of Agriculture, Forestry, and Fisheries of Japan (Rice Genome Project MP-1129) to Y.I. and H.K.

Notes

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: Makoto Matsuoka (pj.ca.u-ayogan.rga.1rgaun@otokam).

W in BoxOnline version contains Web-only data.

Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.105.030981.

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